Methods and materials for assessing rna expression

ABSTRACT

This document provides methods and materials for assessing RNA expression. For example, methods and materials for detecting the presence, absence, or amount of target nucleic acid (e.g., target RNA or target cDNA produced from target RNA), kits for detecting the presence, absence, or amount of target nucleic acid (e.g., target RNA or target cDNA produced from target RNA), and methods for making such kits are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.13/921,322, filed Jun. 19, 2013, which is a continuation of U.S.application Ser. No. 13/027,919, filed Feb. 15, 2011 (now U.S. Pat. No.8,470,533), which claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 61/304,798, filed Feb. 15, 2010. The disclosure ofthe prior application is considered part of (and is incorporated byreference in) the disclosure of this application.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in detecting RNAexpression. For example, this document relates to methods and materialsinvolved in using an enzymatic amplification cascade of restrictionendonucleases to detect RNA expression.

2. Background

The polymerase chain reaction (PCR) is a technique commonly used toamplify and detect nucleic acid. For example, real-time PCR can be usedto amplify and detect small amounts of template DNA present in a sample.Typically, PCR involves adding a nucleic acid primer pair and aheat-stable DNA polymerase, such as Taq polymerase, to a sample believedto contain a targeted nucleic acid to be amplified. Subjecting thesample containing the primer pair and polymerase to a thermal cyclingprocess sets in motion a chain reaction in which the targeted DNAtemplate located between the primers is exponentially amplified. Thepresence of this amplified template can be detected using techniquessuch as gel electrophoresis or the amount of amplified template can beassessed using techniques such as those involving the use offluorescently labeled probes. In some cases, reverse transcriptaseenzymes can be used with PCR to detect or quantify RNA expression withina sample.

SUMMARY

This document provides methods and materials for detecting RNAexpression. For example, this document relates to methods and materialsinvolved in using an enzymatic amplification cascade of restrictionendonucleases to detect RNA expression. Information about RNA expressionthat occurs within a cell of an organism can be important forunderstanding that organism's health and/or susceptibilities to certaindiseases or disorders. For example, elevated or reduced RNA expressionof certain nucleic acids in a human's genome can indicate that thatparticular human is susceptible to developing a certain disease.

In some cases, this document provides methods and materials fordetecting target nucleic acid (e.g., target RNA or target cDNA producedfrom target RNA). For example, this document provides methods andmaterials for detecting the presence, absence, or amount of targetnucleic acid (e.g., target RNA or target cDNA produced from target RNA),kits for detecting the presence, absence, or amount of target nucleicacid (e.g., target RNA or target cDNA produced from target RNA), andmethods for making such kits.

In general, the methods and materials provided herein can includeperforming an enzymatic amplification cascade of restrictionendonucleases as described herein to detect target nucleic acid (e.g.,target RNA or target cDNA produced from target RNA) in a manner that israpid, inexpensive, sensitive, and specific. For example, a sample(e.g., an RNA sample from a cell or tissue) can be obtained from anorganism (e.g., a human) and/or processed such that target nucleic acid,if present within the sample, is capable of hybridizing to probe nucleicacid of an enzymatic amplification cascade of restriction endonucleasesdescribed herein. In some cases, such an obtained and/or processedsample can be assessed for the presence, absence, or amount of targetnucleic acid using an enzymatic amplification cascade of restrictionendonucleases described herein without using a nucleic acidamplification technique (e.g., a PCR-based nucleic acid technique).Assessing samples (e.g., biological samples) for the presence, absence,or amount of target nucleic acid using an enzymatic amplificationcascade of restriction endonucleases described herein without using anucleic acid amplification technique can allow patients as well asmedical, laboratory, or veterinarian personnel (e.g., clinicians,physicians, physician's assistants, laboratory technicians, researchscientists, and veterinarians) to assess RNA expression using a nucleicacid-based assay without the need for potentially expensive thermalcycling devices and potentially time consuming thermal cyclingtechniques. In addition, the methods and materials provided herein canallow patients as well as medical, laboratory, or veterinarian personnelto assess expression of any type of RNA suspected of being presentwithin an organism (e.g., a mammal such as a human). For example, themethods and materials provided herein can be used to detect the presenceor absence of expression of particular mRNA within a human.

In general, one aspect of this document features a method for assessingRNA expression. The method comprises, or consists essentially of, (a)contacting a sample to be assessed for expression of a target RNA with aprobe nucleic acid comprising an amplifying restriction endonuclease anda nucleotide sequence complementary to a sequence of the target RNA or acDNA of the target RNA under conditions wherein, if the target RNA orthe cDNA is present in the sample, at least a portion of the target RNAor the cDNA hybridizes to at least a portion of the probe nucleic acidto form a double-stranded portion of nucleic acid comprising arestriction endonuclease cut site, (b) contacting the double-strandedportion of nucleic acid with a recognition restriction endonucleasehaving the ability to cut the double-stranded portion of nucleic acid atthe restriction endonuclease cut site under conditions wherein therecognition restriction endonuclease cleaves the double-stranded portionof nucleic acid at the restriction endonuclease cut site, therebyseparating a portion of the probe nucleic acid comprising the amplifyingrestriction endonuclease from at least another portion of the probenucleic acid, (c) contacting the portion of the probe nucleic acidcomprising the amplifying restriction endonuclease with a reporternucleic acid comprising a double-stranded portion of nucleic acidcomprising a restriction endonuclease cut site of the amplifyingrestriction endonuclease under conditions wherein the amplifyingrestriction endonuclease cleaves the reporter nucleic acid at therestriction endonuclease cut site of the amplifying restrictionendonuclease, thereby separating a portion of the reporter nucleic acidfrom at least another portion of the reporter nucleic acid, and (d)determining the presence or absence of the portion of the reporternucleic acid, wherein the presence of the portion of the reporternucleic acid indicates that the sample contains the target RNA or thecDNA, thereby indicating expression of the target RNA, and wherein theabsence of the portion of the reporter nucleic acid indicates that thesample does not contain the target RNA or the cDNA, thereby indicating alack of expression of the target RNA. The sample can be a sampleobtained from a mammal. The sample can be a sample obtained from ahuman. The sample can be a sample obtained from an organism selectedfrom the group consisting of bovine, porcine, and equine species. Thesample can be a sample obtained from a plant. The probe nucleic acid cancomprise the nucleotide sequence complementary to a sequence of thetarget RNA. The probe nucleic acid can comprise the nucleotide sequencecomplementary to a sequence of the cDNA. The target RNA can be an mRNA.The target RNA can be an rRNA. The target RNA can be a tRNA. The targetRNA can be an mRNA that encodes a polypeptide selected from the groupconsisting of interleukin polypeptides, enzymatic polypeptides, andstructural polypeptides. The sample can be selected from the groupconsisting of blood samples, skin samples, tissue samples, and tumorsamples. Prior to step (a), the sample can be a sample that wasprocessed to remove non-nucleic acid material from the sample, therebyincreasing the concentration of nucleic acid, if present, within thesample. The sample can be a sample that was subjected to an RNAextraction technique. Prior to step (a), the sample can be a sample thatwas subjected to a nucleic acid amplification technique to increase theconcentration of one or more nucleic acids, if present, within thesample. The sample can be a sample that was subjected to a PCR-basedtechnique designed to amplify the target nucleic acid. Prior to step(a), the method can comprise removing non-nucleic acid material from thesample, thereby increasing the concentration of nucleic acid, ifpresent, within the sample. The removing can comprise performing an RNAextraction technique. Prior to step (a), the method can compriseperforming a nucleic acid amplification technique to increase theconcentration of one or more nucleic acids, if present, within thesample. The nucleic acid amplification technique can comprise aPCR-based technique designed to amplify the cDNA. Prior to step (a), themethod can comprise removing non-nucleic acid material from the sample,thereby increasing the concentration of nucleic acid, if present, withinthe sample, and performing a reverse transcriptase reaction to generatethe cDNA, if present, within the sample. The probe nucleic acid can besingle-stranded probe nucleic acid. The probe nucleic acid can beattached to a solid support. The probe nucleic acid can be directlyattached to a solid support. The portion of the probe nucleic acidcomprising the amplifying restriction endonuclease can be released fromthe solid support via the step (b). Step (a) and step (b) can beperformed in the same compartment, or step (a), step (b), and step (c)can be performed in the same compartment, or step (a), step (b), step(c), and step (d) can be performed in the same compartment. Step (a) andstep (b) can be performed in a first compartment, and step (c) can beperformed in a second compartment. Step (a) and step (b) can beperformed by adding the sample to a compartment comprising the probenucleic acid and the recognition restriction endonuclease. The probenucleic acid can comprise (i) a single-stranded portion comprising thenucleotide sequence complementary to the sequence of the target RNA orthe cDNA and (ii) a double-stranded portion. The probe nucleic acid cancomprise a first nucleic acid strand comprising the nucleotide sequencecomplementary to the sequence of the target RNA or the cDNA hybridizedto a second nucleic acid strand comprising the amplifying restrictionendonuclease. The first nucleic acid strand can be attached to a solidsupport. The first nucleic acid strand can be directly attached to asolid support. A portion of the second nucleic acid strand can hybridizewith the first nucleic acid strand to form the double-stranded portion.The portion of the probe nucleic acid comprising the amplifyingrestriction endonuclease that is separated from the at least anotherportion of the probe nucleic acid in step (b) can comprise a portion ofthe first nucleic acid strand and all of the second strand. The portionof the probe nucleic acid comprising the amplifying restrictionendonuclease that is separated from the at least another portion of theprobe nucleic acid in step (b) can comprise at least a portion of thetarget RNA or the cDNA.

In some cases, the method can comprise using a plurality of the probenucleic acid in the step (a). The method can comprise using a pluralityof the reporter nucleic acid in the step (c). The reporter nucleic acidin the step (c) can be in molar excess of the portion of the probenucleic acid comprising the amplifying restriction endonuclease from thestep (b). The number of molecules of the portion of the probe nucleicacid comprising the amplifying restriction endonuclease that isseparated from the at least another portion of the probe nucleic acid instep (b) can be in an essentially linear relationship to the number ofmolecules of the target RNA or the cDNA present in the sample. Thereporter nucleic acid can be attached to a solid support. The reporternucleic acid can be directly attached to a solid support. The reporternucleic acid can comprise a single-stranded portion of nucleic acid. Thereporter nucleic acid can comprise a label. The label can be afluorescent label, a radioactive label, an enzyme label, or a redoxlabel. The portion of the reporter nucleic acid that is separated fromthe at least another portion of the reporter nucleic acid can comprisethe label. The reporter nucleic acid can comprise a first nucleic acidstrand comprising the label hybridized to a second nucleic acid strand.The second nucleic acid strand can be attached to a solid support. Thesecond nucleic acid strand can be directly attached to a solid support.A portion of the first nucleic acid strand can hybridize with the secondnucleic acid strand to form the double-stranded portion of nucleic acidcomprising the restriction endonuclease cut site of the amplifyingrestriction endonuclease. The reporter nucleic acid can comprise a thirdnucleic acid strand. The third nucleic acid strand can hybridize withthe second nucleic acid strand to form the double-stranded portion ofnucleic acid comprising the restriction endonuclease cut site of theamplifying restriction endonuclease. The reporter nucleic acid can beattached to a solid support, and the portion of the reporter nucleicacid that is separated from the at least another portion of the reporternucleic acid and that comprises the label can be released from the solidsupport via the step (c). The determining step (d) can comprisedetecting the label. The label can be a fluorescent label, and thedetermining step (d) can comprise detecting the fluorescent label. Thedetermining step (d) can comprise detecting the portion of the reporternucleic acid separated from the at least another portion of the reporternucleic acid using a capillary electrophoresis technique. Steps (a),(b), and (c) can be performed without nucleic acid amplification, orsteps (a), (b), (c), and (d) can be performed without nucleic acidamplification. The determining step can comprise determining the amountof the target RNA or the cDNA present within the sample.

In another aspect, this document features a method for assessing RNAexpression. The method comprises, or consists essentially of, (a)contacting a sample to be assessed for expression of a target RNA with aprobe nucleic acid comprising an initial amplifying restrictionendonuclease and a nucleotide sequence complementary to a sequence ofthe target RNA or a cDNA of the target RNA under conditions wherein, ifthe target RNA or the cDNA is present in the sample, at least a portionof the target RNA or the cDNA hybridizes to at least a portion of theprobe nucleic acid to form a double-stranded portion of nucleic acidcomprising a restriction endonuclease cut site, (b) contacting thedouble-stranded portion of nucleic acid with a recognition restrictionendonuclease having the ability to cut the double-stranded portion ofnucleic acid at the restriction endonuclease cut site under conditionswherein the recognition restriction endonuclease cleaves thedouble-stranded portion of nucleic acid at the restriction endonucleasecut site, thereby separating a portion of the probe nucleic acidcomprising the initial amplifying restriction endonuclease from at leastanother portion of the probe nucleic acid, (c) contacting the portion ofthe probe nucleic acid comprising the initial amplifying restrictionendonuclease with a first nucleic acid comprising a secondary amplifyingrestriction endonuclease and a double-stranded portion of nucleic acidcomprising a restriction endonuclease cut site of the initial amplifyingrestriction endonuclease under conditions wherein the initial amplifyingrestriction endonuclease cleaves the first nucleic acid at therestriction endonuclease cut site of the initial amplifying restrictionendonuclease, thereby separating a portion of the first nucleic acidcomprising the secondary amplifying restriction endonuclease from atleast another portion of the first nucleic acid, (d) contacting theportion of the first nucleic acid comprising the secondary amplifyingrestriction endonuclease with a second nucleic acid comprising theinitial amplifying restriction endonuclease and a double-strandedportion of nucleic acid comprising a restriction endonuclease cut siteof the secondary amplifying restriction endonuclease under conditionswherein the secondary amplifying restriction endonuclease cleaves thesecond nucleic acid at the restriction endonuclease cut site of thesecondary amplifying restriction endonuclease, thereby separating aportion of the second nucleic acid comprising the initial amplifyingrestriction endonuclease from at least another portion of the secondnucleic acid, (e) contacting the portion of the second nucleic acidcomprising the initial amplifying restriction endonuclease with areporter nucleic acid comprising a double-stranded portion of nucleicacid comprising a restriction endonuclease cut site of the initialamplifying restriction endonuclease under conditions wherein the initialamplifying restriction endonuclease cleaves the reporter nucleic acid atthe restriction endonuclease cut site of the initial amplifyingrestriction endonuclease, thereby separating a portion of the reporternucleic acid from at least another portion of the reporter nucleic acid,and (f) determining the presence or absence of the portion of thereporter nucleic acid, wherein the presence of the portion of thereporter nucleic acid indicates that the sample contains the target RNAor the cDNA, thereby indicating expression of the target RNA, andwherein the absence of the portion of the reporter nucleic acidindicates that the sample does not contain the target RNA or the cDNA,thereby indicating a lack of expression of the target RNA. The samplecan be a sample obtained from a mammal. The sample can be a sampleobtained from a human. The sample can be a sample obtained from anorganism selected from the group consisting of bovine, porcine, andequine species. The sample can be a sample obtained from a plant. Theprobe nucleic acid can comprise the nucleotide sequence complementary toa sequence of the target RNA. The probe nucleic acid can comprise thenucleotide sequence complementary to a sequence of the cDNA. The targetRNA can be an mRNA. The target RNA can be an rRNA. The target RNA can bea tRNA. The target RNA can be an mRNA that encodes a polypeptideselected from the group consisting of interleukin polypeptides,enzymatic polypeptides, and structural polypeptides. The sample can beselected from the group consisting of blood samples, skin samples,tissue samples, and tumor samples. Prior to step (a), the sample can bea sample that was processed to remove non-nucleic acid material from thesample, thereby increasing the concentration of nucleic acid, ifpresent, within the sample. The sample can be a sample that wassubjected to an RNA extraction technique. Prior to step (a), the samplecan be a sample that was subjected to a nucleic acid amplificationtechnique to increase the concentration of one or more nucleic acids, ifpresent, within the sample. The sample can be a sample that wassubjected to a PCR-based technique designed to amplify the targetnucleic acid. Prior to step (a), the method can comprise removingnon-nucleic acid material from the sample, thereby increasing theconcentration of nucleic acid, if present, within the sample. Theremoving can comprise performing an RNA extraction technique. Prior tostep (a), the method can comprise performing a nucleic acidamplification technique to increase the concentration of one or morenucleic acids, if present, within the sample. The nucleic acidamplification technique can comprise a PCR-based technique designed toamplify the cDNA. Prior to step (a), the method can comprise removingnon-nucleic acid material from the sample, thereby increasing theconcentration of nucleic acid, if present, within the sample, andperforming a reverse transcriptase reaction to generate the cDNA, ifpresent, within the sample. The probe nucleic acid can besingle-stranded probe nucleic acid. The probe nucleic acid can beattached to a solid support. The probe nucleic acid can be directlyattached to a solid support. The portion of the probe nucleic acidcomprising the initial amplifying restriction endonuclease can bereleased from the solid support via the step (b). Step (a) and step (b)can be performed in the same compartment, step (a), step (b), and step(c) can be performed in the same compartment, step (a), step (b), step(c), and step (d) can be performed in the same compartment, step (a),step (b), step (c), step (d), and step (e) can be performed in the samecompartment, or step (a), step (b), step (c), step (d), step (e), andstep (f) can be performed in the same compartment. Step (c) and step (d)can be performed in the same compartment. Step (a) and step (b) can beperformed in a first compartment, and step (c) and step (d) can beperformed in a second compartment. Step (a) and step (b) can beperformed by adding the sample to a compartment comprising the probenucleic acid and the recognition restriction endonuclease. Step (c) andstep (d) can be performed by adding the portion of the probe nucleicacid comprising the initial amplifying restriction endonuclease to acompartment comprising the first nucleic acid and the second nucleicacid. The probe nucleic acid can comprise (i) a single-stranded portioncomprising the nucleotide sequence complementary to the sequence of thetarget RNA or the cDNA and (ii) a double-stranded portion. The probenucleic acid can comprise a first nucleic acid strand comprising thenucleotide sequence complementary to the sequence of the target RNA orthe cDNA hybridized to a second nucleic acid strand comprising theinitial amplifying restriction endonuclease. The first nucleic acidstrand can be attached to a solid support. The first nucleic acid strandcan be directly attached to a solid support. A portion of the secondnucleic acid strand can hybridize with the first nucleic acid strand toform the double-stranded portion. The portion of the probe nucleic acidcomprising the initial amplifying restriction endonuclease that isseparated from the at least another portion of the probe nucleic acid instep (b) can comprise a portion of the first nucleic acid strand and allof the second strand. The portion of the probe nucleic acid comprisingthe initial amplifying restriction endonuclease that is separated fromthe at least another portion of the probe nucleic acid in step (b) cancomprise at least a portion of the target RNA or the cDNA.

In some cases, the method can comprise using a plurality of the probenucleic acid in the step (a). The method can comprise using a pluralityof the reporter nucleic acid in the step (e). The reporter nucleic acidin the step (e) can be in molar excess of the portion of the probenucleic acid comprising the initial amplifying restriction endonucleasefrom the step (b). The number of molecules of the portion of the probenucleic acid comprising the initial amplifying restriction endonucleasethat is separated from the at least another portion of the probe nucleicacid in step (b) can be in an essentially linear relationship to thenumber of molecules of the target RNA or the cDNA present in the sample.The first nucleic acid and the second nucleic acid can be attached to asolid support. The first nucleic acid and the second nucleic acid can bedirectly attached to a solid support. The first nucleic acid and thesecond nucleic acid can be attached to a solid support in the samecompartment. The portion of the first nucleic acid comprising thesecondary amplifying restriction endonuclease can be released from thesolid support via the step (c). The portion of the second nucleic acidcomprising the initial amplifying restriction endonuclease can bereleased from the solid support via the step (d). The first nucleic acidcan comprise a first nucleic acid strand comprising the secondaryamplifying restriction endonuclease hybridized to a second nucleic acidstrand to form the double-stranded portion of nucleic acid comprisingthe restriction endonuclease cut site of the initial amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. The second nucleic acid cancomprise a first nucleic acid strand comprising the initial amplifyingrestriction endonuclease hybridized to a second nucleic acid strand toform the double-stranded portion of nucleic acid comprising therestriction endonuclease cut site of the secondary amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. The reporter nucleic acid can beattached to a solid support. The reporter nucleic acid can be directlyattached to a solid support. The reporter nucleic acid can comprise asingle-stranded portion of nucleic acid. The reporter nucleic acid cancomprise a label. The label can be a fluorescent label, a radioactivelabel, an enzyme label, or a redox label. The portion of the reporternucleic acid that is separated from the at least another portion of thereporter nucleic acid can comprise the label. The reporter nucleic acidcan comprise a first nucleic acid strand comprising the label hybridizedto a second nucleic acid strand. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. A portion of the first nucleicacid strand can hybridize with the second nucleic acid strand to formthe double-stranded portion of nucleic acid comprising the restrictionendonuclease cut site of the initial amplifying restrictionendonuclease. The reporter nucleic acid can comprise a third nucleicacid strand. The third nucleic acid strand can hybridize with the secondnucleic acid strand to form the double-stranded portion of nucleic acidcomprising the restriction endonuclease cut site of the initialamplifying restriction endonuclease. The reporter nucleic acid can beattached to a solid support, and the portion of the reporter nucleicacid that is separated from the at least another portion of the reporternucleic acid and that comprises the label can be released from the solidsupport via the step (e). The determining step (f) can comprisedetecting the label. The label can be a fluorescent label, and thedetermining step (f) comprises detecting the fluorescent label. Thedetermining step (f) can comprise detecting the portion of the reporternucleic acid separated from the at least another portion of the reporternucleic acid using a capillary electrophoresis technique. Steps (a),(b), (c), (d), and (e) can be performed without nucleic acidamplification, or steps (a), (b), (c), (d), (e), and (f) can beperformed without nucleic acid amplification. The determining step cancomprise determining the amount of the target RNA or the cDNA presentwithin the sample.

In another aspect, this document features a method for assessing RNAexpression. The method comprises, or consists essentially of, (a)contacting a sample to be assessed for expression of a target RNA with aprobe nucleic acid comprising an initial amplifying restrictionendonuclease and a nucleotide sequence complementary to a sequence ofthe target RNA or a cDNA of the target RNA under conditions wherein, ifthe target RNA or the cDNA is present in the sample, at least a portionof the target RNA or the cDNA hybridizes to at least a portion of theprobe nucleic acid to form a double-stranded portion of nucleic acidcomprising a restriction endonuclease cut site, (b) contacting thedouble-stranded portion of nucleic acid with a recognition restrictionendonuclease having the ability to cut the double-stranded portion ofnucleic acid at the restriction endonuclease cut site under conditionswherein the recognition restriction endonuclease cleaves thedouble-stranded portion of nucleic acid at the restriction endonucleasecut site, thereby separating a portion of the probe nucleic acidcomprising the initial amplifying restriction endonuclease from at leastanother portion of the probe nucleic acid, (c) contacting the portion ofthe probe nucleic acid comprising the initial amplifying restrictionendonuclease with a first reporter nucleic acid comprising a secondaryamplifying restriction endonuclease and a double-stranded portion ofnucleic acid comprising a restriction endonuclease cut site of theinitial amplifying restriction endonuclease under conditions wherein theinitial amplifying restriction endonuclease cleaves the first reporternucleic acid at the restriction endonuclease cut site of the initialamplifying restriction endonuclease, thereby separating a portion of thefirst nucleic acid comprising the secondary amplifying restrictionendonuclease from at least another portion of the first nucleic acid,(d) contacting the portion of the first reporter nucleic acid comprisingthe secondary amplifying restriction endonuclease with a second reporternucleic acid comprising the initial amplifying restriction endonucleaseand a double-stranded portion of nucleic acid comprising a restrictionendonuclease cut site of the secondary amplifying restrictionendonuclease under conditions wherein the initial amplifying restrictionendonuclease cleaves the second nucleic acid at the restrictionendonuclease cut site of the secondary amplifying restrictionendonuclease, thereby separating a portion of the second nucleic acidcomprising the initial amplifying restriction endonuclease from at leastanother portion of the second nucleic acid, and (e) determining thepresence or absence of the portion of the first reporter nucleic acid,the second reporter nucleic acid, or both the first reporter nucleicacid and the second reporter nucleic acid, wherein the presenceindicates that the sample contains the target RNA or the cDNA, therebyindicating expression of the target RNA, and wherein the absenceindicates that the sample does not contain the target RNA or the cDNA,thereby indicating a lack of expression of the target RNA. The samplecan be a sample obtained from a mammal. The sample can be a sampleobtained from a human. The sample can be a sample obtained from anorganism selected from the group consisting of bovine, porcine, andequine species. The sample can be a sample obtained from a plant. Theprobe nucleic acid can comprise the nucleotide sequence complementary toa sequence of the target RNA. The probe nucleic acid can comprise thenucleotide sequence complementary to a sequence of the cDNA. The targetRNA can be an mRNA. The target RNA can be an rRNA. The target RNA can bea tRNA. The target RNA can be an mRNA that encodes a polypeptideselected from the group consisting of interleukin polypeptides,enzymatic polypeptides, and structural polypeptides. The sample can beselected from the group consisting of blood samples, skin samples,tissue samples, and tumor samples. Prior to step (a), the sample can bea sample that was processed to remove non-nucleic acid material from thesample, thereby increasing the concentration of nucleic acid, ifpresent, within the sample. The sample can be a sample that wassubjected to an RNA extraction technique. Prior to step (a), the samplecan be a sample that was subjected to a nucleic acid amplificationtechnique to increase the concentration of one or more nucleic acids, ifpresent, within the sample. The sample can be a sample that wassubjected to a PCR-based technique designed to amplify the targetnucleic acid. Prior to step (a), the method can comprise removingnon-nucleic acid material from the sample, thereby increasing theconcentration of nucleic acid, if present, within the sample. Theremoving can comprise performing an RNA extraction technique. Prior tostep (a), the method can comprise performing a nucleic acidamplification technique to increase the concentration of one or morenucleic acids, if present, within the sample. The nucleic acidamplification technique can comprise a PCR-based technique designed toamplify the cDNA. Prior to step (a), the method can comprise removingnon-nucleic acid material from the sample, thereby increasing theconcentration of nucleic acid, if present, within the sample, andperforming a reverse transcriptase reaction to generate the cDNA, ifpresent, within the sample. The probe nucleic acid can besingle-stranded probe nucleic acid. The probe nucleic acid can beattached to a solid support. The probe nucleic acid can be directlyattached to a solid support. The portion of the probe nucleic acidcomprising the initial amplifying restriction endonuclease can bereleased from the solid support via the step (b). Step (a) and step (b)can be performed in the same compartment, step (a), step (b), and step(c) can be performed in the same compartment, step (a), step (b), step(c), and step (d) can be performed in the same compartment, or step (a),step (b), step (c), step (d), and step (e) can be performed in the samecompartment. Step (c) and step (d) can be performed in the samecompartment. Step (a) and step (b) can be performed in a firstcompartment, and step (c) and step (d) can be performed in a secondcompartment. Step (a) and step (b) can be performed by adding the sampleto a compartment comprising the probe nucleic acid and the recognitionrestriction endonuclease. Step (c) and step (d) can be performed byadding the portion of the probe nucleic acid comprising the initialamplifying restriction endonuclease to a compartment comprising thefirst reporter nucleic acid and the second reporter nucleic acid. Theprobe nucleic acid can comprise (i) a single-stranded portion comprisingthe nucleotide sequence complementary to the sequence of the target RNAor the cDNA and (ii) a double-stranded portion. The probe nucleic acidcan comprise a first nucleic acid strand comprising the nucleotidesequence complementary to the sequence of the target RNA or the cDNAhybridized to a second nucleic acid strand comprising the initialamplifying restriction endonuclease. The first nucleic acid strand canbe attached to a solid support. The first nucleic acid strand can bedirectly attached to a solid support. A portion of the second nucleicacid strand can hybridize with the first nucleic acid strand to form thedouble-stranded portion. The portion of the probe nucleic acidcomprising the initial amplifying restriction endonuclease that isseparated from the at least another portion of the probe nucleic acid instep (b) can comprise a portion of the first nucleic acid strand and allof the second strand. The portion of the probe nucleic acid comprisingthe initial amplifying restriction endonuclease that is separated fromthe at least another portion of the probe nucleic acid in step (b) cancomprise at least a portion of the target RNA or the cDNA.

In some cases, the method can comprise using a plurality of the probenucleic acid in the step (a). The method can comprise using a pluralityof the first reporter nucleic acid in the step (c). The first reporternucleic acid in the step (c) can be in molar excess of the portion ofthe probe nucleic acid comprising the initial amplifying restrictionendonuclease from the step (b). The method can comprise using aplurality of the second reporter nucleic acid in the step (d). Thesecond reporter nucleic acid in the step (d) can be in molar excess ofthe portion of the probe nucleic acid comprising the initial amplifyingrestriction endonuclease from the step (b). The number of molecules ofthe portion of the probe nucleic acid comprising the initial amplifyingrestriction endonuclease that is separated from the at least anotherportion of the probe nucleic acid in step (b) can be in an essentiallylinear relationship to the number of molecules of the target RNA or thecDNA present in the sample. The first reporter nucleic acid and thesecond reporter nucleic acid can be attached to a solid support. Thefirst reporter nucleic acid and the second reporter nucleic acid can bedirectly attached to a solid support. The first reporter nucleic acidand the second reporter nucleic acid can be attached to a solid supportin the same compartment. The portion of the first reporter nucleic acidcomprising the secondary amplifying restriction endonuclease can bereleased from the solid support via the step (c). The portion of thesecond reporter nucleic acid comprising the initial amplifyingrestriction endonuclease can be released from the solid support via thestep (d). The first reporter nucleic acid can comprise a label. Thelabel can be a fluorescent label, a radioactive label, an enzyme label,or a redox label. The second reporter nucleic acid can comprise a label.The label can be a fluorescent label, a radioactive label, an enzymelabel, or a redox label. The first reporter nucleic acid and the secondreporter nucleic acid can comprise a label. The first reporter nucleicacid and the second reporter nucleic acid can comprise the same label.The label can be a fluorescent label, a radioactive label, an enzymelabel, or a redox label. The first reporter nucleic acid can be attachedto a solid support, the portion of the first reporter nucleic acid thatis separated from the at least another portion of the first reporternucleic acid can comprise a label, and the portion of the first reporternucleic acid that is separated from the at least another portion of thefirst reporter nucleic acid and that comprises the label can be releasedfrom the solid support via the step (c). The first reporter nucleic acidcan comprise a first nucleic acid strand comprising the secondaryamplifying restriction endonuclease hybridized to a second nucleic acidstrand to form the double-stranded portion of nucleic acid comprisingthe restriction endonuclease cut site of the initial amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. The first nucleic acid strand cancomprise a label. The label can be a fluorescent label, a radioactivelabel, an enzyme label, or a redox label. The second nucleic acid strandcan comprise a label. The label can be a fluorescent label, aradioactive label, an enzyme label, or a redox label. The secondreporter nucleic acid can be attached to a solid support, the portion ofthe second reporter nucleic acid that is separated from the at leastanother portion of the second reporter nucleic acid can comprise alabel, and the portion of the second reporter nucleic acid that isseparated from the at least another portion of the second reporternucleic acid and that comprises the label can be released from the solidsupport via the step (d). The second reporter nucleic acid can comprisea first nucleic acid strand comprising the initial amplifyingrestriction endonuclease hybridized to a second nucleic acid strand toform the double-stranded portion of nucleic acid comprising therestriction endonuclease cut site of the secondary amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. The first nucleic acid strand cancomprise a label. The label can be a fluorescent label, a radioactivelabel, an enzyme label, or a redox label. The second nucleic acid strandcan comprise a label. The label can be a fluorescent label, aradioactive label, an enzyme label, or a redox label. The portion of thefirst reporter nucleic acid separated from the at least another portionof the first reporter nucleic acid can comprise a fluorescent label, theportion of the second reporter nucleic acid separated from the at leastanother portion of the second reporter nucleic acid can comprise afluorescent label, and the determining step (e) can comprise detectingthe fluorescent label. The determining step (e) can comprise detectingthe portion of the first reporter nucleic acid separated from the atleast another portion of the first reporter nucleic acid using acapillary electrophoresis technique. The determining step (e) cancomprise detecting the portion of the second reporter nucleic acidseparated from the at least another portion of the second reporternucleic acid using a capillary electrophoresis technique. Steps (a),(b), (c), and (d) can be performed without nucleic acid amplification,or steps (a), (b), (c), (d), and (e) can be performed without nucleicacid amplification. The determining step can comprise determining theamount of the target RNA or the cDNA present within the sample.

In another aspect, this document features a kit for assessing RNAexpression. The kit comprises, or consists essentially of, a probenucleic acid comprising an amplifying restriction endonuclease and anucleotide sequence complementary to a sequence of a target RNA or acDNA of the target RNA, wherein at least a portion of the target RNA orthe cDNA is capable of hybridizing to at least a portion of the probenucleic acid to form a double-stranded portion of nucleic acidcomprising a restriction endonuclease cut site. The probe nucleic acidcan be single-stranded probe nucleic acid. The kit can comprise a solidsupport, and the probe nucleic acid can be attached to the solidsupport. A portion of the probe nucleic acid comprising the amplifyingrestriction endonuclease can be releasable from the solid support viacleavage with a recognition restriction endonuclease having the abilityto cleave at the restriction endonuclease cut site. The kit can furthercomprise the recognition restriction endonuclease. The probe nucleicacid can comprise (i) a single-stranded portion comprising thenucleotide sequence complementary to the sequence of the target RNA orthe cDNA and (ii) a double-stranded portion. The probe nucleic acid cancomprise a first nucleic acid strand comprising the nucleotide sequencecomplementary to the sequence of the target RNA or the cDNA hybridizedto a second nucleic acid strand comprising the amplifying restrictionendonuclease. The kit can further comprise a reporter nucleic acidcomprising a double-stranded portion of nucleic acid comprising arestriction endonuclease cut site of the amplifying restrictionendonuclease. The kit can comprise a solid support, and the reporternucleic acid can be attached to the solid support. The reporter nucleicacid can be directly attached to the solid support. The reporter nucleicacid can comprise a single-stranded portion of nucleic acid. Thereporter nucleic acid can comprise a label. The label can be afluorescent label, a radioactive label, an enzyme label, or a redoxlabel. A portion of the reporter nucleic acid comprising the label canbe capable of being separated from at least another portion of thereporter nucleic acid via cleavage by the amplifying restrictionendonuclease. The reporter nucleic acid can comprise a first nucleicacid strand comprising the label hybridized to a second nucleic acidstrand. The kit can further comprise: (a) a first signal expansionnucleic acid comprising a secondary amplifying restriction endonucleaseand a double-stranded section having a restriction endonuclease cut sitefor the amplifying restriction endonuclease, and (b) a second signalexpansion nucleic acid comprising the amplifying restrictionendonuclease and a double-stranded section having a restrictionendonuclease cut site for the secondary amplifying restrictionendonuclease. The probe nucleic acid can be lyophilized. All theingredients of the kit can be lyophilized or dry.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting an exemplary method for detecting targetnucleic acid using probe nucleic acid, a recognition restrictionendonuclease, and reporter nucleic acid.

FIG. 2 is a schematic of an exemplary configuration of probe nucleicacid that can be used with the methods and materials provided herein fordetecting target nucleic acid.

FIG. 3 is a schematic depicting an exemplary method for detecting targetnucleic acid using probe nucleic acid, a recognition restrictionendonuclease, first signal expansion nucleic acid, second signalexpansion nucleic acid, and reporter nucleic acid.

FIG. 4 is a schematic of an exemplary configuration of first signalexpansion nucleic acid and second signal expansion nucleic acid that canbe used with the methods and materials provided herein for detectingtarget nucleic acid. Such first signal expansion nucleic acid and secondsignal expansion nucleic acid can be used with or without reporternucleic acid. When used without a separate reporter nucleic acid step,such signal expansion nucleic acid can be referred to as reporternucleic acid.

FIG. 5 is a schematic of an exemplary configuration of first signalexpansion nucleic acid and second signal expansion nucleic acid that canbe used with the methods and materials provided herein for detectingtarget nucleic acid. Such first signal expansion nucleic acid and secondsignal expansion nucleic acid can be used with or without reporternucleic acid. When used without a separate reporter nucleic acid step,such signal expansion nucleic acid can be referred to as reporternucleic acid.

FIG. 6 contains line graphs demonstrating the effect of targetoligonucleotide concentration (A) and recognition restrictionendonuclease concentration (B) on the cleavage of HRP-labeled nucleicacid as detected by the formation of colored reaction product.

FIG. 7 is a schematic of an exemplary configuration for a single-use,pen-style point of care device.

DETAILED DESCRIPTION

This document provides methods and materials for detecting RNAexpression. For example, this document provides methods and materialsfor detecting the presence, absence, or amount of target nucleic acid(e.g., target RNA or target cDNA produced from target RNA) in a samplefrom an organism, kits for detecting the presence, absence, or amount oftarget nucleic acid (e.g., target RNA or target cDNA produced fromtarget RNA) in a sample from an organism, and methods for making suchkits.

Any type of organism (e.g., plant or animal) can be assessed using themethods and materials provided herein to determine the presence,absence, or amount of expression of a target RNA. Examples of organismsthat can be assessed using the methods and materials provided herein todetermine the presence, absence, or amount of expression of a target RNAinclude, without limitation, plants (e.g., trees, flowers, shrubs,grains, grasses, and legumes), mammals (e.g., humans, dogs, cats, cows,horses, pigs, sheep, goats, monkeys, buffalo, bears, whales, anddolphins), avian species (e.g., chickens, turkeys, ostrich, emus,cranes, and falcons), and non-mammalian animals (e.g., mollusks, frogs,lizards, snakes, and insects). For example, human tissue can be assessedto determine the amount of expression of a particular RNA target.

Any type of biological sample can be used with the methods and materialsprovided herein to assess RNA expression. For example, any type ofbiological sample that is obtained from an organism to be tested andthat contains the organism's RNA (e.g., mRNA, tRNA, or rRNA) or cDNAproduced from the organism's RNA can be used as described herein.Examples of samples that can be used as described herein include,without limitation, blood samples, skin samples, tissue samples (e.g.,tissue biopsy samples), and tumor samples.

The methods and materials provided herein can be used to assess anorganism or a tissue of an organism for expression of any type of RNA.Examples of possible RNA that can be assessed using the methods andmaterials provided herein include, without limitation, mRNA, tRNA, andrRNA. In some cases, an mRNA that has an expression level (e.g., anincreased expression level or a decreased expression level) that isassociated with the presence or absence of a particular disease ordisorder, an increased or decreased susceptibility to a particulardisease or disorder, or a favorable or unfavorable outcome of aparticular disease or disorder can be assessed using the methods andmaterials provided herein. In some cases, expression of a target RNAlisted in Table 1 in an organism or a tissue of an organism listed inTable 1 can be assessed using the methods and materials provided herein.When designing a method for detecting a target RNA listed in Table 1,probe nucleic acid can be designed that is complementary to a portion ofany of the indicated sequences from Table 1.

TABLE 1 Examples of target RNA that can be assessed in the indicatedtissues. Over- expression, Under- expression, Possible Organism/TissuePresence, or Associated or Cell Type RNA Target Absence Condition Humanlung IGF-binding proteins Over- poor adenocarcinoma (IGFBPs), IGFBP-2expression prognostics RNA Human prostate prostaglandin E receptor Over-resistance cancer cells EP4 subtype (EP4) RNA expression progressionHuman liver RNA encoding Over- Infection, biopsy interferon-inducibleexpression fibrosis genes progression Human DOCK8 RNA Under- Shorterhepatocellular expression patient carcinoma survival Human colorectalRNA encoding cyclo- expression Cancer cancer oxygenase-2 (COX-2)recurrence Human ovarian RNA encoding Elafin Over- Poor carcinomaexpression prognosis Human nasal RNA viruses: Over- influenza swabinfluenza A and B expression Human liver RNA virus: Over- Infection,biopsy Hepatitis C virus expression liver damage

In one embodiment, a method for assessing RNA expression can includedetermining whether or not a biological sample (e.g., a tissue sample)obtained from an organism contains a target nucleic acid of interest(e.g., a target RNA or target cDNA produced from target RNA). Forexample, a biological sample (e.g., a blood sample to be tested) can beplaced in contact with probe nucleic acid. The probe nucleic acid can bedesigned to have a single-stranded portion with a nucleotide sequencethat is complementary to at least a portion of the target nucleic acidto be detected. In this case, target nucleic acid present within thesample can hybridize with the complementary sequence of thissingle-stranded portion of the probe nucleic acid to form adouble-stranded section with one strand being target nucleic acid andthe other strand being probe nucleic acid. In addition, thesingle-stranded portion of the probe nucleic acid having the nucleotidesequence that is complementary to at least a portion of the targetnucleic acid to be detected can be designed such that hybridization withthe target nucleic acid creates a restriction endonuclease cut site.Thus, target nucleic acid present within the sample can hybridize withthe complementary sequence of the single-stranded portion of the probenucleic acid to form a double-stranded section that creates a cut sitefor a restriction endonuclease. This cut site created by thehybridization of target nucleic acid to probe nucleic acid can bereferred to as a recognition restriction endonuclease cut site. Inaddition, a restriction endonuclease that cleaves nucleic acid at such arecognition restriction endonuclease cut site can be referred to as arecognition restriction endonuclease.

The probe nucleic acid also can be designed to contain a restrictionendonuclease. This restriction endonuclease, which can be a component ofthe probe nucleic acid, can be referred to as an amplifying restrictionendonuclease. An amplifying restriction endonuclease is typically adifferent restriction endonuclease than the restriction endonucleasethat is used as a recognition restriction endonuclease. For example,when an EcoRI restriction endonuclease is used as a recognitionrestriction endonuclease, a restriction endonuclease other than an EcoRIrestriction endonuclease (e.g., an Hind III restriction endonuclease) isused as an amplifying restriction endonuclease. Thus, in general, probenucleic acid is designed to contain an amplifying restrictionendonuclease and to have a nucleotide sequence such that the targetnucleic acid can hybridize to the probe nucleic acid and create arecognition restriction endonuclease cut site for a recognitionrestriction endonuclease. In some cases, the probe nucleic acid can beattached to a solid support (e.g., a well of a microtiter plate). Forexample, the probe nucleic acid can be attached to a solid support suchthat cleavage at the recognition restriction endonuclease cut site viathe recognition restriction endonuclease releases a portion of the probenucleic acid that contains the amplifying restriction endonuclease.

After contacting the sample (e.g., a biological sample) that may or maynot contain target nucleic acid with the probe nucleic acid that isattached to a solid support, the target nucleic acid, if present in thesample, can hybridize to the probe nucleic acid and create therecognition restriction endonuclease cut site. At this point, therecognition restriction endonuclease, whether added to the reaction oralready present in the reaction, can cleave the probe nucleic acid atthe recognition restriction endonuclease cut sites that are formed bythe hybridization of target nucleic acid to the probe nucleic acid,thereby releasing the portion of the probe nucleic acid that containsthe amplifying restriction endonuclease from the solid support. Thenumber of amplifying restriction endonuclease-containing portions of theprobe nucleic acid that are released from the solid support can be in anessentially linear relationship (e.g., essentially a one-for-onerelationship) with the number of target nucleic acid molecules thathybridize with the probe nucleic acid to form the recognitionrestriction endonuclease cut site.

The portions of the probe nucleic acid containing the amplifyingrestriction endonuclease that were released from the solid support canbe collected and placed in contact with reporter nucleic acid. Forexample, the released portions of the probe nucleic acid, if present,can be transferred from one well of a microtiter plate (e.g., a 96-wellplate) that contained the probe nucleic acid to another well of amicrotiter plate that contains the reporter nucleic acid. The reporternucleic acid can be designed to have a double-stranded portion with arestriction endonuclease cut site for the amplifying restrictionendonuclease of the probe nucleic acid. This restriction endonucleasecut site for the amplifying restriction endonuclease can be referred toas an amplifying restriction endonuclease cut site. If portions of theprobe nucleic acid containing the amplifying restriction endonucleaseare present and placed in contact with the reporter nucleic acid, thenthe reporter nucleic acid can be cleaved at the amplifying restrictionendonuclease cut site by the amplifying restriction endonuclease. Sincethe amplifying restriction endonucleases of the released portions of theprobe nucleic acid are free to carry out repeated cleavage events, thenumber of reporter nucleic acid molecules that are cleaved can greatlyexceed the number of amplifying restriction endonucleases present in thereaction. For example, the number of cleaved reporter nucleic acidmolecules can greatly exceed (e.g., exponentially exceed) the number ofamplifying restriction endonucleases present in the reaction andtherefore can greatly exceed (e.g., exponentially exceed) the number oftarget nucleic acid molecules that were present in the sample contactedwith the probe nucleic acid. Such a greatly expanded relationship (e.g.,an exponential relationship) can allow very small amounts of targetnucleic acid present in the sample to be readily detected.

After the released portions of the probe nucleic acid, if present, arecontacted with the reporter nucleic acid, the presence or absence ofcleaved reporter nucleic acid can be determined. The presence of cleavedreporter nucleic acid can indicate that the sample contained the targetnucleic acid, thereby indicating that cells within the sample expressedthe target RNA for which the sample is being tested, while the absenceof cleaved reporter nucleic acid can indicate that the sample lacked thetarget nucleic acid, thereby indicating that cells within the sample didnot express the target RNA for which the sample is being tested. In somecases, the amount of cleaved reporter nucleic acid can be determined. Insuch cases, the amount of cleaved reporter nucleic acid can indicate theamount of target nucleic acid present in the sample, which can indicatethe relative amount of expression of the target RNA for which the sampleis being tested. A standard curve using known amounts of target nucleicacid can be used to aid in the determination of the amount of targetnucleic acid present within a sample. For example, increasing knownamounts of target RNA or target cDNA corresponding to the target RNA canbe included in an assay to aid in the quantification of target RNAwithin a sample being tested.

In some cases, the reporter nucleic acid can contain a label to aid inthe detection of cleaved reporter nucleic acid. For example, reporternucleic acid can contain a fluorescent label and a quencher such thatcleaved reporter nucleic acid provides a fluorescent signal anduncleaved reporter nucleic acid does not provide a fluorescent signal.In some cases, the reporter nucleic acid can contain a label (e.g., acolorimetric label, a fluorescent label or an enzyme (e.g., a redoxenzyme) such as horse radish peroxidase) and can be attached to a solidsupport (e.g., a well of a microtiter plate). For example, the reporternucleic acid can be attached to a solid support such that cleavage atthe amplifying restriction endonuclease cut site by the amplifyingrestriction endonuclease releases a portion of the reporter nucleic acidthat contains the label. The resulting reaction mixture can be collectedand assessed for the presence, absence, or amount of released portionsof the reporter nucleic acid using the label. For example, the releasedportions of the reporter nucleic acid, if present, can be transferredfrom one well of a microtiter plate (e.g., a 96-well plate) thatcontained the reporter nucleic acid to another well of a microtiterplate, where the transferred material can be assessed for a signal fromthe label.

One example of a method of detecting target nucleic acid that includesusing probe nucleic acid and reporter nucleic acid is set forth inFIG. 1. With reference to FIG. 1, first reaction chamber 100 (e.g., amicrotiter plate well) can contain probe nucleic acid 101. Probe nucleicacid 101 can be attached (e.g., immobilized) to solid support 102 andcan include amplifying restriction endonuclease 103 (Ra). Probe nucleicacid 101 can be attached to solid support 102 such that amplifyingrestriction endonuclease 103 is released from solid support 102 uponcleavage of a nucleic acid component of probe nucleic acid 101. Probenucleic acid 101 can have a single-stranded section having a nucleotidesequence that is complementary to at least a portion of target nucleicacid 104. Probe nucleic acid 101 can be contacted with a sample that mayor may not contain target nucleic acid 104. If target nucleic acid 104is present, at least a portion of target nucleic acid 104 and probenucleic acid 101 can hybridize to form a double-stranded section ofnucleic acid. Such a double-stranded section can contain at least onerecognition restriction endonuclease cut site 105. Addition ofrecognition restriction endonuclease 106 (Rr) to first reaction chamber100 can result in the cleave of probe nucleic acid 101 at recognitionrestriction endonuclease cut site 105 formed by one strand of probenucleic acid and one strand of target nucleic acid, thereby releasingportion 107 of probe nucleic acid 101 from solid support 102. Portion107 can include amplifying restriction endonuclease 103.

The reaction product from first reaction chamber 100 containing releasedportion 107, if target nucleic acid 104 was present, can be transferred(e.g., manually or automatically) to second reaction chamber 120. Secondreaction chamber 120 can contain reporter nucleic acid 121. Reporternucleic acid 121 can be attached (e.g., immobilized) to solid support122 and can include marker (e.g., a label) 123 (M). Reporter nucleicacid 121 can be attached to solid support 122 such that marker 123 isreleased from solid support 122 upon cleavage of a nucleic acidcomponent of reporter nucleic acid 121. Reporter nucleic acid 121 canhave at least one double-stranded portion that contains at least oneamplifying restriction endonuclease cut site 124. Addition of thereaction product from first reaction chamber 100 to second reactionchamber 120 can result in the cleavage of reporter nucleic acid 121 atamplifying restriction endonuclease cut site 124 if the reaction productcontains portion 107. Such cleavage of reporter nucleic acid 121 canresult in the release of portion 127 from solid support 122. Portion 127can include marker 123.

The reaction product from second reaction chamber 120 can be assessed todetermine the presence, absence, or amount of portion 127. The presenceof portion 127 can indicate that the sample contained target nucleicacid 104, while the absence of portion 127 can indicate that the samplelacked target nucleic acid 104. In some cases, the amount of portion 127can be determined. In such cases, the amount of portion 127 can indicatethe amount of target nucleic acid 104 present in the sample. Thepresence, absence, or amount of portion 127 can be determined usingmarker 123, and portion 127 having marker 123 can be distinguished fromuncleaved reporter nucleic acid 121 having marker 123 since, in thisexample, portion 127 is released from solid support 122, while uncleavedreporter nucleic acid 121 remains attached to solid support 122. Forexample, in some cases, the reaction product from second reactionchamber 120 can be transferred to third reaction chamber where thepresence or absence of portion 127 via marker 123 is assessed. Ifportion 127 is present, the amount of portion 127 present can bequantified.

Probe nucleic acid 101 and reporter nucleic acid 121 can have variousconfigurations. For example, with reference to FIG. 1, probe nucleicacid 101 can be designed to have a single nucleic acid strand such thatthe entire nucleic acid component of probe nucleic acid 101 issingle-stranded prior to contact with target nucleic acid 104. Inanother example, with reference to FIG. 2, probe nucleic acid 101 can bedesigned to have first strand 128 and second strand 108. First strand128 can be attached to solid support 102 and can be designed to have asingle-stranded section having a nucleotide sequence that iscomplementary to at least a portion of target nucleic acid 104. Secondstrand 108 can include amplifying restriction endonuclease 103 and canhave a single-stranded section having a nucleotide sequence that canhybridize to first strand 128. In some cases, first strand 128 andsecond strand 108 can be synthesized or obtained separately and thenmixed together to form probe nucleic acid 101. For example, first strand128 can be synthesized, biotinylated, and attached to astreptavidin-coated solid support. After synthesizing the nucleic acidcomponent of second strand 108 and attaching amplifying restrictionendonuclease 103 to the synthesized nucleic acid component, secondstrand 108 can be incubated with first strand 128 to form nucleic acidprobe 101. In some cases, probe nucleic acid 101 can contain more thantwo strands. For example, probe nucleic acid can include first strand150, second strand 152, and third strand 154. In this case, first strand150 can be attached to solid support 102, second strand 152 can behybridized to first strand 150 and can include a single-stranded sectionhaving a nucleotide sequence that is complementary to at least a portionof target nucleic acid 104, and third strand 154 can be hybridized tosecond strand 152 and can be attached to amplifying restrictionendonuclease 103. Similar one, two, three, or more strand configurationscan be used to make reporter nucleic acid.

In another embodiment, a method for detecting target nucleic acid caninclude contacting a sample (e.g., a biological sample to be tested)with probe nucleic acid. The probe nucleic acid can be designed to havea single-stranded portion with a nucleotide sequence that iscomplementary to at least a portion of the target nucleic acid to bedetected. In this case, target nucleic acid present within the samplecan hybridize with the complementary sequence of this single-strandedportion of the probe nucleic acid to form a double-stranded section withone strand being target nucleic acid and the other strand being probenucleic acid. In addition, the single-stranded portion of the probenucleic acid having the nucleotide sequence that is complementary to atleast a portion of the target nucleic acid to be detected can bedesigned such that hybridization with the target nucleic acid creates arecognition restriction endonuclease cut site. Thus, target nucleic acidpresent within the sample can hybridize with the complementary sequenceof the single-stranded portion of the probe nucleic acid to form adouble-stranded section that creates a recognition restrictionendonuclease cut site for a recognition restriction endonuclease. Theprobe nucleic acid also can be designed to contain an amplifyingrestriction endonuclease. Since this method includes the use of two ormore different amplifying restriction endonucleases, the amplifyingrestriction endonuclease that is a component of the probe nucleic acidcan be referred to as a first or an initial amplifying restrictionendonuclease, with additional amplifying restriction endonucleases beingreferred to as second, third, and so on or secondary, tertiary, and soon amplifying restriction endonucleases. This initial amplifyingrestriction endonuclease is typically a different restrictionendonuclease than the restriction endonuclease that is used as arecognition restriction endonuclease. For example, when an EcoRIrestriction endonuclease is used as a recognition restrictionendonuclease, a restriction endonuclease other than an EcoRI restrictionendonuclease (e.g., an Hind III restriction endonuclease) is used as aninitial amplifying restriction endonuclease. Thus, in general, probenucleic acid is designed to contain an initial amplifying restrictionendonuclease and to have a nucleotide sequence such that the targetnucleic acid can hybridize to the probe nucleic acid and create arecognition restriction endonuclease cut site for a recognitionrestriction endonuclease. In some cases, the probe nucleic acid can beattached to a solid support (e.g., a well of a microtiter plate). Forexample, the probe nucleic acid can be attached to a solid support suchthat cleavage at the recognition restriction endonuclease cut site viathe recognition restriction endonuclease releases a portion of the probenucleic acid that contains the initial amplifying restrictionendonuclease.

After contacting the sample that may or may not contain target nucleicacid with the probe nucleic acid that is attached to a solid support,the target nucleic acid, if present in the sample, can hybridize to theprobe nucleic acid and create the recognition restriction endonucleasecut site. At this point, the recognition restriction endonuclease,whether added to the reaction or already present in the reaction, cancleave the probe nucleic acid at the recognition restrictionendonuclease cut sites that are formed by the hybridization of targetnucleic acid to the probe nucleic acid, thereby releasing the portion ofthe probe nucleic acid that contains the initial amplifying restrictionendonuclease from the solid support. The number of initial amplifyingrestriction endonuclease-containing portions of the probe nucleic acidthat are released from the solid support can be in an essentially linearrelationship (e.g., essentially a one-for-one relationship) with thenumber of target nucleic acid molecules that hybridize with the probenucleic acid to form the recognition restriction endonuclease cut site.

The portions of the probe nucleic acid containing the initial amplifyingrestriction endonuclease that were released from the solid support canbe collected and placed in contact with first signal expansion nucleicacid and second signal expansion nucleic acid. The first signalexpansion nucleic acid can be designed to have a double-stranded portionwith a restriction endonuclease cut site for the initial amplifyingrestriction endonuclease of the probe nucleic acid. This restrictionendonuclease cut site for the initial amplifying restrictionendonuclease can be referred to as an initial amplifying restrictionendonuclease cut site. The first signal expansion nucleic acid also canbe designed to contain a secondary amplifying restriction endonuclease.The second signal expansion nucleic acid can be designed to have adouble-stranded portion with a restriction endonuclease cut site for thesecondary amplifying restriction endonuclease of the first signalexpansion nucleic acid. This restriction endonuclease cut site for thesecondary amplifying restriction endonuclease can be referred to as asecondary amplifying restriction endonuclease cut site. The secondsignal expansion nucleic acid also can be designed to contain an initialamplifying restriction endonuclease. For example, when an EcoRIrestriction endonuclease is used as a recognition restrictionendonuclease and an HindIII restriction endonuclease is used as aninitial amplifying restriction endonuclease of the probe nucleic acid, aSmaI restriction endonuclease can be used as a secondary amplifyingrestriction endonuclease of the first signal expansion nucleic acid andan HindIII restriction endonuclease can be used as the initialamplifying restriction endonuclease of the second signal expansionnucleic acid.

In some cases, the first signal expansion nucleic acid and second signalexpansion nucleic acid can be attached to a solid support (e.g., a wellof a microtiter plate). For example, the first signal expansion nucleicacid can be attached to a solid support such that cleavage at theinitial amplifying restriction endonuclease cut site via the initialamplifying restriction endonuclease releases a portion of the firstsignal expansion nucleic acid that contains the secondary amplifyingrestriction endonuclease, and the second signal expansion nucleic acidcan be attached to a solid support such that cleavage at the secondaryamplifying restriction endonuclease cut site via the secondaryamplifying restriction endonuclease releases a portion of the secondsignal expansion nucleic acid that contains the initial amplifyingrestriction endonuclease. The first signal expansion nucleic acid can beattached to the same solid support (e.g., two different sub-compartmentsof a larger compartment) that contains the second signal expansionnucleic acid provided that the secondary amplifying restrictionendonuclease of uncleaved first signal expansion nucleic acid is unableto cleave the second signal expansion nucleic acid and provided that theinitial amplifying restriction endonuclease of uncleaved second signalexpansion nucleic acid is unable to cleave the first signal expansionnucleic acid. In some cases, the first signal expansion nucleic acid canbe attached to the same solid support within a joint compartment suchthat the first signal expansion nucleic acid is within a firstcompartment of the joint compartment and the second signal expansionnucleic acid is within a second compartment of the joint compartment. Insuch cases, the secondary amplifying restriction endonuclease ofuncleaved first signal expansion nucleic acid in the first compartmentis unable to cleave the second signal expansion nucleic acid located inthe second compartment, while the secondary amplifying restrictionendonuclease of cleaved first signal expansion nucleic acid is capableof moving (e.g., diffusing) from the first compartment to the secondcompartment to cleave the second signal expansion nucleic acid locatedin the second compartment. In addition, the initial amplifyingrestriction endonuclease of uncleaved second signal expansion nucleicacid in the second compartment is unable to cleave the first signalexpansion nucleic acid located in the first compartment, while theinitial amplifying restriction endonuclease of cleaved second signalexpansion nucleic acid is capable of moving (e.g., diffusing) from thesecond compartment to the first compartment to cleave the first signalexpansion nucleic acid located in the first compartment.

If portions of the probe nucleic acid containing the initial amplifyingrestriction endonuclease are present and placed in contact with thefirst signal expansion nucleic acid, then the first signal expansionnucleic acid can be cleaved at the initial amplifying restrictionendonuclease cut site by the initial amplifying restrictionendonuclease, thereby releasing a portion of the first signal expansionnucleic acid that contains the secondary amplifying restrictionendonuclease from the solid support. The released portions of the firstsignal expansion nucleic acid containing the secondary amplifyingrestriction endonuclease can be free to cleave the second signalexpansion nucleic acid at the secondary amplifying restrictionendonuclease cut site, thereby releasing a portion of the second signalexpansion nucleic acid that contains the initial amplifying restrictionendonuclease from the solid support. Since the initial amplifyingrestriction endonucleases of the released portions of the probe nucleicacid, the initial amplifying restriction endonucleases of the releasedportions of the second signal expansion nucleic acid, and the secondaryamplifying restriction endonucleases of the released portions of thefirst signal expansion nucleic acid are free to carry out repeatedcleavage events, the number of released portions containing the initialamplifying restriction endonucleases is greatly increased from thenumber that were released by the recognition restriction endonuclease.For example, the number of cleaved first signal expansion nucleic acidmolecules can greatly exceed (e.g., exponentially exceed) the number ofreleased portions of the probe nucleic acid, and the number of cleavedsecond signal expansion nucleic acid molecules can greatly exceed (e.g.,exponentially exceed) the number of released portions of the probenucleic acid. Such a greatly expanded relationship (e.g., an exponentialrelationship) can allow very small amounts of target nucleic acidpresent in the sample to be readily detected.

In some cases, this method can be performed with the first signalexpansion nucleic acid being attached to a solid support that isdifferent from the solid support that contains the second signalexpansion nucleic acid. For example, the first signal expansion nucleicacid can be attached to one well of a microtiter plate, while the secondsignal expansion nucleic acid can be attached to a different well of amicrotiter plate. In this case, the resulting reaction material from thewell with the first signal expansion nucleic acid can be collected andtransferred to the well containing the second signal expansion nucleicacid.

The portions of the second signal expansion nucleic acid containing theinitial amplifying restriction endonuclease that were released from thesolid support containing the second signal expansion nucleic acid alongwith any other released portions in this reaction (e.g., the releasedportions of the probe nucleic acid containing the initial amplifyingrestriction endonuclease and the released portions of the first signalexpansion nucleic acid containing the secondary amplifying restrictionendonuclease) can be collected and placed in contact with reporternucleic acid. For example, the released portions, if present, can betransferred from one well of a microtiter plate (e.g., a 96-well plate)that contained the second signal expansion nucleic acid to another wellof a microtiter plate that contains the reporter nucleic acid. Thereporter nucleic acid can be designed to have a double-stranded portionwith a restriction endonuclease cut site for the initial amplifyingrestriction endonuclease. If released portions containing the initialamplifying restriction endonuclease are present and placed in contactwith the reporter nucleic acid, then the reporter nucleic acid can becleaved at the initial amplifying restriction endonuclease cut site bythe initial amplifying restriction endonuclease. Since the initialamplifying restriction endonucleases of the released portions are freeto carry out repeated cleavage events, the number of reporter nucleicacid molecules that are cleaved can greatly exceed the number of initialamplifying restriction endonucleases present in the reaction. Forexample, the number of cleaved reporter nucleic acid molecules cangreatly exceed (e.g., exponentially exceed) the number of initialamplifying restriction endonucleases present in the reaction andtherefore can greatly exceed (e.g., exponentially exceed) the number oftarget nucleic acid molecules that were present in the sample contactedwith the probe nucleic acid. Such a greatly expanded relationship (e.g.,an exponential relationship) can allow very small amounts of targetnucleic acid present in the sample to be readily detected.

After the released portions containing the initial amplifyingrestriction endonuclease, if present, are contacted with the reporternucleic acid, the presence or absence of cleaved reporter nucleic acidcan be determined. The presence of cleaved reporter nucleic acid canindicate that the sample contained the target nucleic acid, therebyindicating that cells within the sample expressed the target RNA forwhich the sample is being tested, while the absence of cleaved reporternucleic acid can indicate that the sample lacked the target nucleicacid, thereby indicating that cells within the sample did not expressthe target RNA for which the sample is being tested.

In some cases, the amount of cleaved reporter nucleic acid can bedetermined. In such cases, the amount of cleaved reporter nucleic acidcan indicate the amount of target nucleic acid present in the sample,which can indicate the relative amount of expression of the target RNAfor which the sample is being tested. A standard curve using knownamounts of target RNA or target cDNA corresponding to the target RNA canbe used to aid in the determination of the amount of expression of thetarget RNA for which that sample is being tested. For example,increasing known amounts of target RNA or target cDNA corresponding tothe target RNA can be included in an assay to aid in the quantificationof target RNA within a sample being tested.

In some cases, the reporter nucleic acid can contain a label to aid inthe detection of cleaved reporter nucleic acid. For example, reporternucleic acid can contain a fluorescent label and a quencher such thatcleaved reporter nucleic acid provides a fluorescent signal anduncleaved reporter nucleic acid does not provide a fluorescent signal.In some cases, the reporter nucleic acid can contain a label (e.g., acolorimetric label, fluorescent label or an enzyme such as horse radishperoxidase) and can be attached to a solid support (e.g., a well of amicrotiter plate). For example, the reporter nucleic acid can beattached to a solid support such that cleavage at the initial amplifyingrestriction endonuclease cut site by the initial amplifying restrictionendonuclease releases a portion of the reporter nucleic acid thatcontains the label. The resulting reaction mixture can be collected andassessed for the presence, absence, or amount of released portions ofthe reporter nucleic acid using the label. For example, the releasedportions of the reporter nucleic acid, if present, can be transferredfrom one well of a microtiter plate (e.g., a 96-well plate) thatcontained the reporter nucleic acid to another well of a microtiterplate, where the transferred material can be assessed for a signal fromthe label.

In some cases, the presence or absence of cleaved first signal expansionnucleic acid, cleaved second signal expansion nucleic acid, or both canbe determined. The presence of such cleaved nucleic acid can indicatethat the sample contained the target nucleic acid, thereby indicatingthat cells within the sample express the target RNA for which the sampleis being tested, while the absence of such cleaved nucleic acid canindicate that the sample lacked the target nucleic acid, therebyindicating that cells within the sample did not express the target RNAfor which the sample is being tested. In some cases, the amount ofcleaved first signal expansion nucleic acid, cleaved second signalexpansion nucleic acid, or both can be determined. In such cases, theamount of cleaved nucleic acid can indicate the amount of expression ofthe target RNA for which the sample is being tested. In these cases, theuse of cleaved first signal expansion nucleic acid, cleaved secondsignal expansion nucleic acid, or both to assess the sample for targetnucleic acid can be in addition to the use of a separate reporternucleic acid step or can replace the use of a separate reporter nucleicacid step. In some cases, the first signal expansion nucleic acid, thesecond signal expansion nucleic acid, or both can be labeled in a mannersimilar to that described herein for the reporter nucleic acid to aid indetection. When the presence, absence, or amount of cleaved first signalexpansion nucleic acid, cleaved second signal expansion nucleic acid, orboth are determined to assess the sample for target nucleic acid, thefirst signal expansion nucleic acid can be referred to as a firstreporter nucleic acid and the second signal expansion nucleic acid canbe referred to as a second reporter nucleic acid even though theyinclude amplifying restriction endonucleases.

A standard curve using known amounts of target RNA or target cDNAcorresponding to the target RNA can be used to aid in the determinationof the amount of expression of the target RNA for which that sample isbeing tested. For example, increasing known amounts of target RNA ortarget cDNA corresponding to the target RNA can be included in an assayto aid in the quantification of target RNA within a sample being testedbased on the amount of cleaved first signal expansion nucleic acid,cleaved second signal expansion nucleic acid, or both.

Examples of a method of detecting target nucleic acid that includesusing probe nucleic acid, first signal expansion nucleic acid, secondsignal expansion nucleic acid, and reporter nucleic acid are set forthin FIGS. 3-5. With reference to FIG. 3, first reaction chamber 200(e.g., a microtiter plate well) can contain probe nucleic acid 201.Probe nucleic acid 201 can be attached (e.g., immobilized) to solidsupport 202 and can include initial amplifying restriction endonuclease203 (Ra). Probe nucleic acid 201 can be attached to solid support 202such that initial amplifying restriction endonuclease 203 is releasedfrom solid support 202 upon cleavage of a nucleic acid component ofprobe nucleic acid 201. Probe nucleic acid 201 can have asingle-stranded section having a nucleotide sequence that iscomplementary to at least a portion of target nucleic acid 204. Probenucleic acid 201 can be contacted with a sample that may or may notcontain target nucleic acid 204. If target nucleic acid 204 is present,at least a portion of target nucleic acid 204 and probe nucleic acid 201can hybridize to form a double-stranded section of nucleic acid. Such adouble-stranded section can contain at least one recognition restrictionendonuclease cut site 205. Addition of recognition restrictionendonuclease 206 (Rr) to first reaction chamber 200 can result in thecleavage of probe nucleic acid 201 at recognition restrictionendonuclease cut site 205 formed by one strand of probe nucleic acid andone strand of target nucleic acid, thereby releasing portion 207 ofprobe nucleic acid 201 from solid support 202. Portion 207 can includeinitial amplifying restriction endonuclease 203.

The reaction product from first reaction chamber 200 containing releasedportion 207, if target nucleic acid 204 was present, can be transferred(e.g., manually or automatically) to second reaction chamber 220. Secondreaction chamber 220 can contain first signal expansion nucleic acid 226and second signal expansion nucleic acid 225. First signal expansionnucleic acid 226 can have at least one double-stranded portion thatcontains at least one initial amplifying restriction endonuclease cutsite 230. First signal expansion nucleic acid 226 can be attached (e.g.,immobilized) to solid support 222 and can include secondary amplifyingrestriction endonuclease 223 (Rb). First signal expansion nucleic acid226 can be attached to solid support 222 such that portion 234containing secondary amplifying restriction endonuclease 223 is releasedfrom solid support 222 upon cleavage of first signal expansion nucleicacid 226 at initial amplifying restriction endonuclease cut site 230.For clarity, frame E of FIG. 3 omits depicting one strand from thecleaved versions of first signal expansion nucleic acid 226 and secondsignal expansion nucleic acid 225.

Second signal expansion nucleic acid 225 can have at least onedouble-stranded portion that contains at least one secondary amplifyingrestriction endonuclease cut site 232. Second signal expansion nucleicacid 225 can be attached (e.g., immobilized) to solid support 222 andcan include initial amplifying restriction endonuclease 224. Secondsignal expansion nucleic acid 225 can be attached to solid support 222such that portion 236 containing initial amplifying restrictionendonuclease 224 is released from solid support 222 upon cleavage ofsecond signal expansion nucleic acid 225 at secondary amplifyingrestriction endonuclease cut site 232. Initial amplifying restrictionendonuclease 203 of probe nucleic acid 201 and initial amplifyingrestriction endonuclease 224 of second signal expansion nucleic acid 225can be the same restriction endonuclease. For example, both can be anEcoRI restriction endonuclease.

Addition of the reaction product from first reaction chamber 200 tosecond reaction chamber 220 can result in the cleavage of first signalexpansion nucleic acid 226 at initial amplifying restrictionendonuclease cut site 230 if the reaction product contains portion 207.Such cleavage of first signal expansion nucleic acid 226 can result inthe release of portion 234 from solid support 222. Portion 234, whichcan include secondary amplifying restriction endonuclease 223, canresult in the cleavage of second signal expansion nucleic acid 225 atsecondary amplifying restriction endonuclease cut site 232. Suchcleavage of second signal expansion nucleic acid 225 can result in therelease of portion 236 from solid support 222. Thus, this reaction canresult in the accumulation of released portions 234 and 236.

The reaction product from second reaction chamber 220 containingreleased portion 207, released portion 234, and released portion 236, iftarget nucleic acid 204 was present, can be transferred (e.g., manuallyor automatically) to third reaction chamber 240. Third reaction chamber240 can contain reporter nucleic acid 241. Reporter nucleic acid 241 canbe attached (e.g., immobilized) to solid support 242 and can includemarker (e.g., a label) 243 (M). Reporter nucleic acid 241 can beattached to solid support 242 such that marker 243 is released fromsolid support 242 upon cleavage of a nucleic acid component of reporternucleic acid 241. Reporter nucleic acid 241 can have at least onedouble-stranded portion that contains at least one initial amplifyingrestriction endonuclease cut site 246. Addition of the reaction productfrom second reaction chamber 220 to third reaction chamber 240 canresult in the cleavage of reporter nucleic acid 241 at initialamplifying restriction endonuclease cut site 246 if the reaction productcontains portion 207 and portion 236. In some cases, reporter nucleicacid 241 can include at least one double-stranded portion that containsat least one cut site for secondary amplifying restriction endonuclease223. In such cases, addition of the reaction product from secondreaction chamber 220 to third reaction chamber 240 can result in thecleavage of reporter nucleic acid 241 at the cut site for secondaryamplifying restriction endonuclease 223 if the reaction product containsportion 234. Cleavage of reporter nucleic acid 241 can result in therelease of portion 247 from solid support 242. Portion 247 can includemarker 243.

The reaction product from third reaction chamber 240 can be assessed todetermine the presence, absence, or amount of portion 247. The presenceof portion 247 can indicate that the sample contained target nucleicacid 204, while the absence of portion 247 can indicate that the samplelacked target nucleic acid 204. In some cases, the amount of portion 247can be determined. In such cases, the amount of portion 247 can indicatethe amount of target nucleic acid 204 present in the sample. Thepresence, absence, or amount of portion 247 can be determined usingmarker 243, and portion 247 having marker 243 can be distinguished fromuncleaved reporter nucleic acid 241 having marker 243 since, in thisexample, portion 247 is released from solid support 242, while uncleavedreporter nucleic acid 241 remains attached to solid support 242. Forexample, in some cases, the reaction product from third reaction chamber24 can be transferred to fourth reaction chamber where the presence orabsence of portion 247 via marker 243 is assessed. If portion 347 ispresent, the amount of portion 247 present can be quantified.

In some cases and with reference to FIGS. 4 and 5, first signalexpansion nucleic acid 226 can include marker (e.g., a label) 243 (M)and second signal expansion nucleic acid 225 can include marker (e.g., alabel) 243 (M). In such cases, cleavage of first signal expansionnucleic acid 226 and cleavage of second signal expansion nucleic acid225 can be assessed using marker 243 to determine the presence, absence,or amount of target nucleic acid within a sample. For example, detector250 can be used to detect marker 243 released from solid support 222.

Probe nucleic acid 201, first signal expansion nucleic acid 226, secondsignal expansion nucleic acid 225, and reporter nucleic acid 241 canhave various configurations. For example, with reference to FIG. 3,probe nucleic acid 201 can be designed to have a single nucleic acidstrand such that the entire nucleic acid component of probe nucleic acid201 is single-stranded prior to contact with target nucleic acid 204. Inanother example, probe nucleic acid 201 can be designed in a manner likeprobe nucleic acid 101 to have two or more strands. See, e.g., FIG. 2.For example, probe nucleic acid 201 can have a first strand and a secondstrand. The first strand can be attached to a solid support and can bedesigned to have a single-stranded section having a nucleotide sequencethat is complementary to at least a portion of target nucleic acid. Thesecond strand can include an initial amplifying restriction endonucleaseand can have a single-stranded section having a nucleotide sequence thatcan hybridize to the first strand. In some cases, the first strand andsecond strand can be synthesized or obtained separately and then mixedtogether to form probe nucleic acid 201. For example, the first strandcan be synthesized, biotinylated, and attached to a streptavidin-coatedsolid support. After synthesizing the nucleic acid component of thesecond strand and attaching an initial amplifying restrictionendonuclease to the synthesized nucleic acid component, the secondstrand can be incubated with the first strand to form nucleic acid probe201. In some cases, probe nucleic acid 201 can contain more than twostrands. For example, probe nucleic acid can include a first strand, asecond strand, and a third strand. In this case, the first strand can beattached to a solid support, the second strand can be hybridized to thefirst strand and can include a single-stranded section having anucleotide sequence that is complementary to at least a portion oftarget nucleic acid, and the third strand can be hybridized to thesecond strand and can be attached to an initial amplifying restrictionendonuclease. Similar one, two, three, or more strand configurations canbe used to make first signal expansion nucleic acid, second signalexpansion nucleic acid, or reporter nucleic acid. For example, firstsignal expansion nucleic acid and second signal expansion nucleic acidcan be designed to have a configuration as shown in FIG. 4 or 5.

Probe nucleic acid described herein typically includes at least onesingle-stranded DNA section that is designed to hybridize with a desiredtarget nucleic acid and thereby create a recognition restrictionendonuclease cut site. The other portions of the probe nucleic acid caninclude DNA, RNA, or other molecules. For example, probe nucleic acidcan include biotin such that the probe nucleic acid can be attached to astreptavidin-coated solid support. In some cases, the single-strandedsection of the probe nucleic acid that is designed to hybridize with adesired target nucleic acid and create a recognition restrictionendonuclease cut site can be RNA or a nucleic acid analog (e.g., apeptide nucleic acid (PNA)) provided that such a single-stranded sectioncan (i) hybridize with the desired target nucleic acid and (ii) create arecognition restriction endonuclease cut site with the complementarytarget nucleic acid sequence that is capable of being cleaved by therecognition restriction endonuclease. Examples of restrictionendonucleases that can be used as recognition restriction endonucleasesto cleave a recognition restriction endonuclease cut site that iscreated between an RNA section of the probe nucleic acid and a DNAsection of the target nucleic acid (e.g., a cDNA produced from an targetRNA) include, without limitation, HhaI, AluI, TaqI, HaeIII, EcoRI,HindII, SalI, and MspI restriction endonucleases.

Probe nucleic acid described herein can be any length provided that thesingle-stranded section of the probe nucleic acid that is designed tohybridize with a desired target nucleic acid is capable of hybridizingto the target nucleic acid and provided that the amplifying restrictionendonuclease of the probe nucleic acid is capable of cleaving itsamplifying restriction endonuclease cut site after the probe nucleicacid is cleaved by a recognition restriction endonuclease. In general,the single-stranded section of the probe nucleic acid that is designedto hybridize with a desired target nucleic acid can be between about 10and about 500 or more nucleotides (e.g., between about 10 and about 400nucleotides, between about 10 and about 300 nucleotides, between about10 and about 200 nucleotides, between about 10 and about 100nucleotides, between about 10 and about 50 nucleotides, between about 10and about 25 nucleotides, between about 20 and about 500 nucleotides,between about 30 and about 500 nucleotides, between about 40 and about500 nucleotides, between about 50 and about 500 nucleotides, betweenabout 15 and about 50 nucleotides, between about 15 and about 25nucleotides, between about 20 and about 50 nucleotides, between about 18and about 25 nucleotides, between about 20 and about 60 nucleotides,between about 25 and about 55 nucleotides, between about 30 and about 50nucleotides, between about 35 and about 45 nucleotides, or between about38 and about 42 nucleotides) in length. The recognition restrictionendonuclease cut site that will be created by the hybridization oftarget nucleic acid to this single-stranded section of the probe nucleicacid can be located at any position alone the single-stranded section.For example, the recognition restriction endonuclease cut site to becreated can be towards the 5′ end, towards the '3 end, or near thecenter of the single-stranded section of the probe nucleic acid. Ingeneral, the overall length of the probe nucleic acid described hereincan be between about 10 and about 2500 or more nucleotides (e.g.,between about 10 and about 2000 nucleotides, between about 10 and about1000 nucleotides, between about 10 and about 500 nucleotides, betweenabout 10 and about 400 nucleotides, between about 10 and about 300nucleotides, between about 10 and about 200 nucleotides, between about10 and about 100 nucleotides, between about 10 and about 50 nucleotides,between about 10 and about 25 nucleotides, between about 20 and about500 nucleotides, between about 30 and about 500 nucleotides, betweenabout 40 and about 500 nucleotides, between about 50 and about 500nucleotides, between about 75 and about 500 nucleotides, between about100 and about 500 nucleotides, between about 150 and about 500nucleotides, between about 15 and about 50 nucleotides, between about 15and about 25 nucleotides, between about 20 and about 50 nucleotides,between about 18 and about 25 nucleotides, between about 20 and about 60nucleotides, between about 25 and about 55 nucleotides, between about 30and about 50 nucleotides, between about 35 and about 45 nucleotides, orbetween about 38 and about 42 nucleotides) in length.

The recognition restriction endonuclease cut site to be created byhybridization of target nucleic acid to the probe nucleic acid can be acut site of any type of restriction endonuclease. In addition, any typeof restriction endonuclease can be used as a recognition restrictionendonuclease to cleave probe nucleic acid upon target nucleic acidhybridization. Examples of restriction endonucleases that can be used asrecognition restriction endonucleases include, without limitation,EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI,HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SphI,StuI, XbaI, AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II, NlaIV,RsaI, TaiI, AasI, BbsI, BseJI, BspTI, ClaI, EcoO109I, I-PpoI, NmuCI,RsrII, TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI,BseMI, BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII,BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI, BsrGI,CspI, Esp3I, KspAI, NsiI, SapI, and TauI restriction endonucleases. Insome cases, nucleic acid encoding a naturally-occurring restrictionendonuclease can be genetically engineered to create a modifiedrestriction endonuclease that has the ability to recognize a particularcut site. Common computer algorithms can be used to locate restrictionendonuclease cut sites along the nucleotide sequence of any desiredtarget nucleic acid. Once located, the sequence of the restrictionendonuclease cut site along with additional flanking sequence (e.g., 5′flanking sequence, 3′ flanking sequence, or both 5′ and 3′ flankingsequence) can be used to design the complementary sequence of the probenucleic acid that is used to hybridize to the target nucleic acid andcreate the recognition restriction endonuclease cut site upon targetnucleic acid hybridization. In some cases, a probe nucleic acid can bedesigned to have the restriction endonuclease cut site located in themiddle or near the middle such that the restriction endonuclease cutsite has both 5′ and 3′ flanking sequences that are complementary to thetarget nucleic acid.

In general, probe nucleic acid can be designed to have a single-strandedsection that is designed to hybridize with desired target nucleic acid(e.g., target RNA or target cDNA produced from target RNA) and to form asingle recognition restriction endonuclease cut site upon target nucleicacid hybridization. In some cases, probe nucleic acid can be designed tohave a single-stranded section that is designed to hybridize withdesired target nucleic acid and to form more than one (e.g., two, three,four, five, six, seven, eight, nine, ten, or more) recognitionrestriction endonuclease cut site upon target nucleic acidhybridization. When more than one recognition restriction endonucleasecut site is used, the multiple recognition restriction endonuclease cutsites can be cut sites for the same restriction endonuclease or cutsites for different restriction endonucleases. For example, probenucleic acid can be designed to have a single-stranded section that isdesigned to hybridize with desired target nucleic acid and to form onerecognition restriction endonuclease cut site for an EcoRI recognitionrestriction endonuclease and one recognition restriction endonucleasecut site for an XbaI recognition restriction endonuclease upon targetnucleic acid hybridization. In such cases, each recognition restrictionendonuclease can be used individually or in combination (e.g., as amixture) to cleave probe nucleic acid that hybridized to target nucleicacid and formed the corresponding recognition restriction endonucleasecut site via such hybridization.

Probe nucleic acid can be designed such that any target nucleic acid(e.g., any target RNA or any target cDNA produced from target RNA) canbe detected. Examples of target nucleic acid that can be detected usingthe methods and materials provided herein include, without limitation,mRNA, tRNA, rRNA, cDNA generated from an RNA, and combinations thereof.When assessing a biological sample for RNA expression, the targetnucleic acid can be an RNA or a cDNA generated from an RNA. Whendetecting an RNA target nucleic acid, restriction endonucleases havingthe ability to cleave a recognition restriction endonuclease cut sitethat is created between a DNA section of the probe nucleic acid and theRNA target nucleic acid can be used as recognition restrictionendonucleases. Examples of such restriction endonucleases include,without limitation, HhaI, AluI, TaqI, HaeIII, EcoRI, HindII, SalI, andMspI restriction endonucleases.

The nucleotide sequence of target nucleic acid to be detected can beobtained from, for example, common nucleic acid sequence databases suchas GenBank®. A portion of target nucleic acid sequence can be selectedusing a computer-based program. For example, a computer-based programcan be used to detect restriction endonuclease cut sites within aportion of target mRNA. Such information can be used to design probenucleic acid such that the single-stranded section creates at least onerecognition restriction endonuclease cut site upon hybridization of thetarget nucleic acid. In some cases, bioinformatics computer-basedprograms and tools can be used to assist in the design of probe nucleicacid. For example, computer programs (e.g., BLAST® and alignmentprograms) and computer databases (e.g., GenBank®) can be used toidentify target RNAs that may be expressed by a particular organism ortissue of an organism. In addition, computer programs such as CLCWorkbench or Vector NTI (Invitrogen) can be used to identify thelocation of restriction endonuclease cut sites within a particularnucleic acid sequence (e.g., a particular target mRNA). In some cases,sequence analysis computer programs can be used to identify sequenceswith limited or an absence of repeats, a presence of high sequencecomplexity of a potential recognition restriction endonuclease cut site,and/or limited or an absence of hairpin structures. Identification ofsuch sequences can help reduce the risk of probe self-hybridization andpotentially unintended cutting by a recognition endonuclease.

Any appropriate method can be used to obtain the nucleic acid componentof the probe nucleic acid. For example, common molecular cloning andchemical nucleic acid synthesis techniques can be used to obtain thenucleic acid component of the probe nucleic acid. In some cases, thenucleic acid component of the probe nucleic acid can be synthesizedusing commercially available automated oligonucleotide synthesizers suchas those available from Applied Biosystems (Foster City, Calif.). Insome cases, probe nucleic acids can be synthesized de novo using any ofa number of procedures widely available in the art. Examples of suchmethods of synthesis include, without limitation, the β-cyanoethylphosphoramidite method (Beaucage et al., Tet. Let., 22:1859-1862 (1981))and the nucleoside H-phosphonate method (Garegg et al., Tet. Let.,27:4051-4054 (1986); Froehler et al., Nucl. Acid Res., 14:5399-5407(1986); Garegg et al., Tet. Let., 27:4055-4058 (1986); and Gaffney etal., Tet. Let., 29:2619-2622 (1988)). These methods can be performed bya variety of commercially-available automated oligonucleotidesynthesizers. In some cases, recombinant nucleic acid techniques such asPCR and those that include using restriction enzyme digestion andligation of existing nucleic acid sequences (e.g., genomic DNA or cDNA)can be used to obtain the nucleic acid component of the probe nucleicacid.

Probe nucleic acid described herein can be attached to a solid support.Examples of solid supports include, without limitation, a well of amicrotiter plate (e.g., a 96-well microtiter plate or ELISA plate),beads (e.g., magnetic, glass, plastic, or gold-coated beads), slides(e.g., glass or gold-coated slides), micro- or nano-particles (e.g.,carbon nanotubes), platinum solid supports, palladium solid supports,and a surface of a chamber or channel within a microfluidic device. Insome cases, a solid support can be a silicon oxide-based solid support,a plastic polymer-based solid support (e.g., a nylon, nitrocellulose, orpolyvinylidene fluoride-based solid support), or a biopolymer-based(e.g., a cross-linked dextran or cellulose-based solid support) solidsupport. Probe nucleic acid can be directly or indirectly attached to asolid support. For example, biotin can be a component of the probenucleic acid, and the probe nucleic acid containing biotin can beindirectly attached to a solid support that is coated with streptavidinvia a biotin-streptavidin interaction. In some cases, probe nucleic acidcan be attached to a solid support via a covalent or non-covalentinteraction. For example, probe nucleic acid can be covalently attachedto magnetic beads as described elsewhere (Albretsen et al., Anal.Biochem., 189(1):40-50 (1990)).

Probe nucleic acid can be designed to contain any type of restrictionendonuclease as an amplifying restriction endonuclease. In general, anamplifying restriction endonuclease of the probe nucleic acid istypically a different restriction endonuclease than the restrictionendonuclease that is used as a recognition restriction endonuclease. Forexample, when an EcoRI restriction endonuclease is used as a recognitionrestriction endonuclease, a restriction endonuclease other than an EcoRIrestriction endonuclease (e.g., an HindIII restriction endonuclease) isused as an amplifying restriction endonuclease. Examples of restrictionendonucleases that can be used as amplifying restriction endonucleasesinclude, without limitation, EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI,HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI,PstI, SacI, SalI, ScaI, SphI, StuI, XbaI, AarI, BanII, BseGI, BspPI,CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI, AasI, BbsI, BseLI, BspTI, ClaI,EcoO109I, I-PpoI, NmuCI, RsrII, TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI,KasI, Acc65I, BbvCI, BseMI, BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI,AccB7I, BbvI, BseMII, BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI,BceAI, BseNI, BsrGI, CspI, Esp3I, KspAI, NsiI, SapI, and TauIrestriction endonucleases. Any number of molecules of the sameamplifying restriction endonuclease can be attached to one probe nucleicacid molecule. For example, a single probe nucleic acid molecule cancontain one, two, three, four, five, or more EcoRI amplifyingrestriction endonuclease molecules. In some cases, a single probenucleic acid molecule can contain two or more (e.g., two, three, four,five, or more) different types of amplifying restriction endonucleases.For example, a single probe nucleic acid molecule can contain threeEcoRI amplifying restriction endonuclease molecules and two BanIIamplifying restriction endonuclease molecules.

Any appropriate method can be used to attach an amplifying restrictionendonuclease to a nucleic acid component of the probe nucleic acid. Insome cases, an amplifying restriction endonuclease can be attached by anionic or covalent attachment. For example, covalent bonds such as amidebonds, disulfide bonds, and thioether bonds, or bonds formed bycrosslinking agents can be used. In some cases, a non-covalent linkagecan be used. The attachment can be a direct attachment or an indirectattachment. For example, a linker can be used to attach an amplifyingrestriction endonuclease to a nucleic acid component of the probenucleic acid. In some cases, nucleic acid can include a thiolmodification, and a restriction endonuclease can be conjugated to thethiol-containing nucleic acid based on succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) using techniquessimilar to those described elsewhere (Dill et al., Biosensors andBioelectronics, 20:736-742 (2004)). In some cases, a biotinylatednucleic acid and a streptavidin-containing restriction endonuclease canbe attached to one another via a biotin-streptavidin interaction. Arestriction endonuclease can be conjugated with streptavidin using, forexample, sulfosuccinimidyl6-(3′-[2-pyridyldithio]-propionamido)hexanoate. An amplifyingrestriction endonuclease can be attached at any location of a nucleicacid component of the probe nucleic acid. For example, an amplifyingrestriction endonuclease can be attached at an end (e.g., a 5′ end or 3′end) of a nucleic acid component, in the middle of a nucleic acidcomponent, or at any position along the length of a nucleic acidcomponent.

Signal expansion nucleic acid (e.g., first signal expansion nucleic acidand second signal expansion nucleic acid) and reporter nucleic aciddescribed herein typically include at least one double-stranded DNAsection that includes an amplifying restriction endonuclease cut site(e.g., an initial amplifying restriction endonuclease cut site, asecondary amplifying restriction endonuclease cut site, or a tertiaryamplifying restriction endonuclease cut site). The other portions of thesignal expansion nucleic acid or reporter nucleic acid can include DNA,RNA, or other molecules. For example, reporter nucleic acid can includebiotin such that the reporter nucleic acid can be attached to astreptavidin-coated solid support. In some cases, one or both strands ofthe double-stranded section of the signal expansion nucleic acid or thereporter nucleic acid that contains an amplifying restrictionendonuclease cut site can be RNA or a nucleic acid analog (e.g., apeptide nucleic acid (PNA)) provided that such a double-stranded sectionis capable of being cleaved by the amplifying restriction endonuclease.Examples of restriction endonucleases that can be used as amplifyingrestriction endonucleases to cleave a DNA:RNA hybrid section of signalexpansion nucleic acid or reporter nucleic acid include, withoutlimitation, HhaI, AluI, TaqI, HaeIII, EcoRI, HindII, SalI, and MspIrestriction endonucleases.

Signal expansion nucleic acid or reporter nucleic acid described hereincan be any length provided that the double-stranded section thatcontains the amplifying restriction endonuclease cut site is capable ofbeing cleaved by the amplifying restriction endonuclease. In general,the double-stranded section of signal expansion nucleic acid or reporternucleic acid can be between about 10 and about 500 or more nucleotides(e.g., between about 10 and about 400 nucleotides, between about 10 andabout 300 nucleotides, between about 10 and about 200 nucleotides,between about 10 and about 100 nucleotides, between about 10 and about50 nucleotides, between about 10 and about 25 nucleotides, between about20 and about 500 nucleotides, between about 30 and about 500nucleotides, between about 40 and about 500 nucleotides, between about50 and about 500 nucleotides, between about 15 and about 50 nucleotides,between about 15 and about 25 nucleotides, between about 20 and about 50nucleotides, or between about 18 and about 25 nucleotides, between about20 and about 60 nucleotides, between about 25 and about 55 nucleotides,between about 30 and about 50 nucleotides, between about 35 and about 45nucleotides, or between about 38 and about 42 nucleotides) in length. Insome cases, the double-stranded section of signal expansion nucleic acidor reporter nucleic acid can be between 5 and 50 nucleotides in length.The amplifying restriction endonuclease cut site of the signal expansionnucleic acid or the reporter nucleic acid can be located at any positionalone the double-stranded section. For example, the amplifyingrestriction endonuclease cut site can be towards the 5′ end, towards the'3 end, or near the center of the double-stranded section of the signalexpansion nucleic acid or the reporter nucleic acid. In general, theoverall length of signal expansion nucleic acid or reporter nucleic aciddescribed herein can be between about 10 and about 2500 or morenucleotides (e.g., between about 10 and about 2000 nucleotides, betweenabout 10 and about 1000 nucleotides, between about 10 and about 500nucleotides, between about 10 and about 400 nucleotides, between about10 and about 300 nucleotides, between about 10 and about 200nucleotides, between about 10 and about 100 nucleotides, between about10 and about 50 nucleotides, between about 10 and about 25 nucleotides,between about 20 and about 500 nucleotides, between about 30 and about500 nucleotides, between about 40 and about 500 nucleotides, betweenabout 50 and about 500 nucleotides, between about 75 and about 500nucleotides, between about 100 and about 500 nucleotides, between about150 and about 500 nucleotides, between about 15 and about 50nucleotides, between about 15 and about 25 nucleotides, between about 20and about 50 nucleotides, between about 18 and about 25 nucleotides,between about 20 and about 60 nucleotides, between about 25 and about 55nucleotides, between about 30 and about 50 nucleotides, between about 35and about 45 nucleotides, or between about 38 and about 42 nucleotides)in length.

The amplifying restriction endonuclease cut site of signal expansionnucleic acid or reporter nucleic acid described herein can be a cut siteof any type of restriction endonuclease. In addition, any type ofrestriction endonuclease can be used as an amplifying restrictionendonuclease to cleave signal expansion nucleic acid or reporter nucleicacid. Examples of restriction endonucleases that can be used asamplifying restriction endonucleases include, without limitation, EcoRI,EcoRII, BamHI, HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI, HaeIII,HgaI, AluI, EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SphI, StuI,XbaI, AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II, NlaIV, RsaI,TaiI, AasI, BbsI, BseJI, BspTI, ClaI, EcoO109I, I-PpoI, NmuCI, RsrII,TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI, BseMI,BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII, BsrFI,Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI, BsrGI, CspI,Esp3I, KspAI, NsiI, SapI, and TauI restriction endonucleases.

In general, signal expansion nucleic acid or reporter nucleic acid canbe designed to have a double-stranded section that contains a singleamplifying restriction endonuclease cut site. In some cases, signalexpansion nucleic acid or reporter nucleic acid provided herein can bedesigned to have a double-stranded section that contains more than one(e.g., two, three, four, five, six, seven, eight, nine, ten, or more)amplifying restriction endonuclease cut site. When more than oneamplifying restriction endonuclease cut site is used, the multipleamplifying restriction endonuclease cut sites can be cut sites for thesame restriction endonuclease or cut sites for different restrictionendonucleases. For example, reporter nucleic acid can be designed tohave a double-stranded section that contains one initial amplifyingrestriction endonuclease cut site for an EcoRI initial amplifyingrestriction endonuclease and one secondary amplifying restrictionendonuclease cut site for an XbaI secondary amplifying restrictionendonuclease.

Any appropriate method can be used to obtain the nucleic acid componentof signal expansion nucleic acid or reporter nucleic acid. For example,common molecular cloning and chemical nucleic acid synthesis techniquescan be used to obtain the nucleic acid component of signal expansionnucleic acid or reporter nucleic acid. In some cases, the nucleic acidcomponent of signal expansion nucleic acid or reporter nucleic acid canbe synthesized using commercially available automated oligonucleotidesynthesizers such as those available from Applied Biosystems (FosterCity, Calif.). In some cases, signal expansion nucleic acid or reporternucleic acid can be synthesized de novo using any of a number ofprocedures widely available in the art. Examples of such methods ofsynthesis include, without limitation, the β-cyanoethyl phosphoramiditemethod (Beaucage et al., Tet. Let., 22:1859-1862 (1981)) and thenucleoside H-phosphonate method (Garegg et al., Tet. Let., 27:4051-4054(1986); Froehler et al., Nucl. Acid Res., 14:5399-5407 (1986); Garegg etal., Tet. Let., 27:4055-4058 (1986); and Gaffney et al., Tet. Let.,29:2619-2622 (1988)). These methods can be performed by a variety ofcommercially-available automated oligonucleotide synthesizers. In somecases, recombinant nucleic acid techniques such as PCR and those thatinclude using restriction enzyme digestion and ligation of existingnucleic acid sequences (e.g., genomic DNA or cDNA) can be used to obtainthe nucleic acid component of signal expansion nucleic acid or reporternucleic acid.

Signal expansion nucleic acid or reporter nucleic acid described hereincan be attached to a solid support. Examples of solid supports include,without limitation, a well of a microtiter plate (e.g., a 96-wellmicrotiter plate or ELISA plate), beads (e.g., magnetic, glass, plastic,or gold-coated beads), slides (e.g., glass or gold-coated slides),micro- or nano-particles (e.g., carbon nanotubes), platinum solidsupports, palladium solid supports, and a surface of a chamber orchannel within a microfluidic device. In some cases, a solid support canbe a silicon oxide-based solid support, a plastic polymer-based solidsupport (e.g., a nylon, nitrocellulose, or polyvinylidene fluoride-basedsolid support) or a biopolymer-based (e.g., a cross-linked dextran orcellulose-based solid support) solid support.

Signal expansion nucleic acid or reporter nucleic acid can be directlyor indirectly attached to a solid support. For example, biotin can be acomponent of signal expansion nucleic acid or reporter nucleic acid, andthe signal expansion nucleic acid or the reporter nucleic acidcontaining biotin can be indirectly attached to a solid support that iscoated with streptavidin via a biotin-streptavidin interaction. In somecases, signal expansion nucleic acid or reporter nucleic acid can beattached to a solid support via a covalent or non-covalent interaction.For example, signal expansion nucleic acid or reporter nucleic acid canbe covalently attached to magnetic beads as described elsewhere(Albretsen et al., Anal. Biochem., 189(1):40-50 (1990)).

Signal expansion nucleic acid can be designed to contain any type ofrestriction endonuclease as an amplifying restriction endonuclease(e.g., an initial amplifying restriction endonuclease, a secondaryamplifying restriction endonuclease, or a tertiary amplifyingrestriction endonuclease). In general, an amplifying restrictionendonuclease of signal expansion nucleic acid is typically a differentrestriction endonuclease than the restriction endonuclease that is usedas a recognition restriction endonuclease. For example, when an EcoRIrestriction endonuclease is used as a recognition restrictionendonuclease, a restriction endonuclease other than an EcoRI restrictionendonuclease (e.g., a HeaIII restriction endonuclease) is used as anamplifying restriction endonuclease. Examples of restrictionendonucleases that can be used as amplifying restriction endonucleasesinclude, without limitation, EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI,HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI,PstI, SacI, SalI, ScaI, SphI, StuI, XbaI, AarI, BanII, BseGI, BspPI,CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI, AasI, BbsI, BseLI, BspTI, ClaI,EcoO109I, I-PpoI, NmuCI, RsrII, TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI,KasI, Acc65I, BbvCI, BseMI, BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI,AccB7I, BbvI, BseMII, BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI,BceAI, BseNI, BsrGI, CspI, Esp3I, KspAI, NsiI, SapI, and TauIrestriction endonucleases. Any number of molecules of the sameamplifying restriction endonuclease can be attached to one signalexpansion nucleic acid molecule. For example, a single signal expansionnucleic acid molecule can contain one, two, three, four, five, or moreEcoRI amplifying restriction endonuclease molecules. In some cases, asingle signal expansion nucleic acid molecule can contain two or more(e.g., two, three, four, five, or more) different types of amplifyingrestriction endonucleases. For example, a single signal expansionnucleic acid molecule can contain three BanII amplifying restrictionendonuclease molecules and two SacII amplifying restriction endonucleasemolecules.

Reporter nucleic acid can be designed to contain a label to aid in thedetection of cleaved reporter nucleic acid. In some cases, signalexpansion nucleic acid can be designed to contain a label. In suchcases, signal expansion nucleic acid containing a label can be used inaddition to reporter nucleic acid or in place of reporter nucleic acidto detect target nucleic acid. Examples of labels that can be acomponent of reporter nucleic acid or signal expansion nucleic acidinclude, without limitation, fluorescent labels (with or without the useof quenchers), dyes, antibodies, radioactive material, enzymes (e.g.,horse radish peroxidase, alkaline phosphatase, laccase, galactosidase,or luciferase), redox labels (e.g., ferrocene redox labels), metallicparticles (e.g., gold nanoparticles), and green fluorescentprotein-based labels. In some cases, for a redox label, such asferrocene, the detector can be an electrode for amperometric assay ofredox molecules. For example, if the redox label is present in a reducedform of ferrocene, then the electrode at high electrode potential canprovide an oxidation of the reduced form of ferrocene, therebyconverting it to an oxidized form of ferrocene. The generated currentcan be proportional to the concentration of ferrocene label in thesolution.

In one embodiment, reporter nucleic acid or signal expansion nucleicacid can contain a fluorescent label and a quencher such that cleavedreporter nucleic acid provides a fluorescent signal and uncleavedreporter nucleic acid does not provide a fluorescent signal. In somecases, the reporter nucleic acid or signal expansion nucleic acid cancontain a label (e.g., a fluorescent label or an enzyme such as horseradish peroxidase) and can be attached to a solid support (e.g., a wellof a microtiter plate). For example, the reporter nucleic acid or signalexpansion nucleic acid can be attached to a solid support such thatcleavage at the amplifying restriction endonuclease cut site by theamplifying restriction endonuclease releases a portion of the reporternucleic acid or the signal expansion nucleic acid that contains thelabel. The resulting reaction mixture can be collected and assessed forthe presence, absence, or amount of released portions of the reporternucleic acid or signal expansion nucleic acid using the label. Forexample, the released portions of the reporter nucleic acid or thesignal expansion nucleic acid, if present, can be transferred from onewell of a microtiter plate (e.g., a 96-well plate) that contained thereporter nucleic acid or the signal expansion nucleic acid to anotherwell of a microtiter plate, where the transferred material can beassessed for a signal from the label. Any number of molecules of a labelcan be attached to one reporter nucleic acid molecule or one signalexpansion nucleic acid molecule. For example, a reporter nucleic acidmolecule or a single signal expansion nucleic acid molecule can containone, two, three, four, five, or more fluorescent molecules.

Any appropriate method can be used to attach a label to a nucleic acidcomponent of reporter nucleic acid or signal expansion nucleic acid. Insome cases, a label can be attached by an ionic or covalent attachment.For example, covalent bonds such as amide bonds, disulfide bonds, andthioether bonds, or bonds formed by crosslinking agents can be used. Insome cases, a non-covalent linkage can be used. The attachment can be adirect attachment or an indirect attachment. For example, a linker canbe used to attach a label to a nucleic acid component of reporternucleic acid or signal expansion nucleic acid. In some cases, nucleicacid can include a thiol modification, and a label can be conjugated tothe thiol-containing nucleic acid based on succinimidyl4[N-maleimidomethyl]cyclo-hexane-1-carboxylate (SMCC) using techniquessimilar to those described elsewhere (Dill et al., Biosensors andBioelectronics, 20:736-742 (2004)). In some cases, a biotinylatednucleic acid and a streptavidin-containing label can be attached to oneanother via a biotin-streptavidin interaction. A label can be conjugatedwith streptavidin using, for example, sulfosuccinimidyl6-(3′[2-pyridyldithio]-propionamido)hexanoate. A label can be attachedat any location of a nucleic acid component of reporter nucleic acid orsignal expansion nucleic acid. For example, a label can be attached atan end (e.g., a 5′ end or 3′ end) of a nucleic acid component, in themiddle of a nucleic acid component, or at any position along the lengthof a nucleic acid component of reporter nucleic acid or signal expansionnucleic acid.

As described herein, the methods and materials provided herein can beused to assess RNA expression in any type of sample (e.g., a biologicalsample). For example, a blood sample or tumor biopsy sample can becollected from a mammal and assessed for target RNA or target cDNAgenerated from target RNA to determine the presence, absence, or amountof target RNA expression within the cells of the sample being tested.Once obtained, a sample to be assessed can be processed to obtainnucleic acid. For example, a nucleic acid extraction can be performed ona blood sample to obtain a sample that is enriched for nucleic acid. Insome cases, a sample can be heated or treated with a cell lysis agent torelease nucleic acid from cells present in the sample.

As described herein, a sample (e.g., a biological sample) can beassessed for the presence, absence, or amount of target nucleic acid(e.g., target RNA or target cDNA generated from target RNA). Whenassessing a sample for target RNA based on the formation of DNA probenucleic acid:RNA target nucleic acid hybrids, the sample can becollected, used, or stored in a manner to preserve RNA from degradation.For example, a sample can be placed or maintained in an Ambion®RNAlater® solution. In some cases, when assessing a sample for an RNAtarget, the sample being tested can be treated with a reversetranscriptase enzyme to produce cDNA from any RNA present within thesample. In such cases, the methods described herein can be performedusing probe nucleic acid designed to hybridize to the produced cDNA ifpresent within the sample. When assessing samples for RNA expression andusing reverse transcriptase to produce cDNA target nucleic acid, thesample can be processed such that the sample contains RNA at arelatively high degree of purity.

In some cases, RNA expression can be assessed using an enzymaticamplification cascade of restriction endonucleases described hereinwithout using a nucleic acid amplification technique (e.g., a PCR-basednucleic acid technique). Assessing samples (e.g., biological samples)for the presence, absence, or amount of expression of target RNA usingan enzymatic amplification cascade of restriction endonucleasesdescribed herein without using a nucleic acid amplification techniquecan allow patients as well as medical, laboratory, or veterinarianpersonnel (e.g., clinicians, physicians, physician's assistants,laboratory technicians, research scientists, and veterinarians) toassess RNA expression without the need for potentially expensive thermalcycling devices and potentially time consuming thermal cyclingtechniques. In some cases, the methods and materials provided herein canbe used in combination with a PCR-based nucleic acid technique. Forexample, reverse transcriptase enzymes can be used to generate cDNA fromRNA present within a sample, and a PCR-based nucleic acid technique canbe performed to amplify nucleic acid (e.g., a target cDNA). Theresulting amplification material can be assessed using an enzymaticamplification cascade of restriction endonucleases described herein todetect the presence, absence, or amount of a particular target nucleicacid (e.g., amplified target cDNA generated from a target RNA presentwithin the sample being tested). In some cases, a limited PCR-basednucleic acid technique can be performed to amplify a target nucleic acidto a point where the amount of amplified target nucleic acid isincreased only slightly over the amount of target nucleic acidoriginally present within the biological sample. For example, a two totwelve cycle PCR technique (e.g., a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 cycle PCR technique) can be performed to slightly increase the amountof amplified target nucleic acid as compared to the amount ofunamplified target nucleic acid originally present (e.g., originallypresent after the use of reverse transcriptase enzymes to generate cDNAfrom RNA present within the sample being tested) within the biologicalsample. Such limited PCR-based nucleic acid techniques, when used incombination with an enzymatic amplification cascade of restrictionendonucleases described herein, can allow medical, laboratory, orveterinarian personnel to assess RNA expression with a potentiallyincreased level of sensitivity and/or specificity without thepotentially lengthy time involved in thermal cycling techniques thatinclude a greater number of cycles. This increased level of sensitivityand/or specificity can be over the high level of sensitivity andspecificity of a comparable testing procedure that includes an enzymaticamplification cascade of restriction endonucleases described hereinwithout the limited PCR-based nucleic acid technique. In some cases, thePCR-based nucleic acid technique can be performed to amplify a targetnucleic acid to a point where the amount of amplified target nucleicacid is easily detectable (e.g., visually detectable using gelelectrophoresis and ethidium bromide staining) For example, a 15 or morecycle PCR technique (e.g., a 20 cycle PCR technique) can be performed toproduce at least ng amounts (e.g., greater than 1 ng, 10 ng, 100 ng, 1μg, 10 μg, or more) of amplified nucleic acid. Such PCR-based nucleicacid techniques, when used in combination with an enzymaticamplification cascade of restriction endonucleases described herein, canallow medical, laboratory, or veterinarian personnel to assess RNAexpression with a potentially increased level of sensitivity and/orspecificity. This increased level of sensitivity and/or specificity canbe over the high level of sensitivity and specificity of a comparabletesting procedure that includes an enzymatic amplification cascade ofrestriction endonucleases described herein without the PCR-based nucleicacid technique.

In some cases, a sample (e.g. a biological sample) can be obtained andsubjected to a culturing technique. For example, a cell sample can beobtained and cultured with medium (e.g., enrichment medium) to enrichthe sample such that the number of cells present in the sample canincrease. Examples of enrichment media include, without limitation,Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM),Iscove's Modified Dulbecco's Media (IMDM), and AIM V® Medium. In somecases, the culture medium can contain a nutrient (e.g. serum such asfetal calf serum), ingredient, or drug that prevents certain cells fromdividing while allowing other cells to divide. In some cases, theculturing technique can include incubating a sample at an appropriatetemperature (e.g. between 15° C. and 45° C., between 20° C. and 45° C.,between 25° C. and 45° C., between 30° C. and 45° C., between 30° C. and40° C., between 35° C. and 45° C., or between 35° C. and 40° C.) for anappropriate period of time (e.g., between about 0.5 hours and 48 hours,between about 0.5 hours and 36 hours, between about 0.5 hours and 24hours, between about 0.5 hours and 12 hours, between about 0.5 hours and8 hours, between about 0.5 hours and 6 hours, between about 0.5 hoursand 5 hours, between about 0.5 hours and 4 hours, between about 0.5hours and 3 hours, between about 0.5 hours and 2 hours, between about 1hour and 4 hours, or between about 2 hours and 4 hours). For example, asample can be obtained and cultured in tissue culture medium at 37° C.for 24-48 hours. Examples of tissue culture techniques that can be usedas described herein include, without limitation, those describedelsewhere (Animal Cell Culture: A Practical Approach, 3rd edition, J.Masters, ed., Oxford University Press, 2000, 336 pp).

In some cases, a sample, obtained and subjected to a culturing techniqueor not, can be processed, for example, to remove non-nucleic acidmaterial, to disrupt cell membranes to release nucleic acid, and/or tocollect or extract nucleic acid, such that nucleic acid of the sample,if present within the sample, is available for hybridization to probenucleic acid. For example, a blood sample or tissue sample can betreated with a lysis buffer and subjected to nucleic acid extractionsuch that a major component of the sample is nucleic acid (e.g., RNA).In some cases, a sample can be homogenized and treated to disrupt cellsthat are present in the sample. For example, a blood sample can besubjected to high speed mechanical homogenization withglass/silica/zirconium/stainless steel beads, can be subjected to hightemperature (e.g., boiling), can be subjected to chemical lysis withdetergents and/or surfactants (e.g., sodium dodecyl sulfate,cetyltrimethylammonium bromide, or sodium lauroyl sarcosin), can besubjected to one or more freeze-thaw cycles using, e.g., liquid nitrogenor dry ice, can be subjected to sonication, or can be subjected tocombinations thereof.

In some cases, a sample treated to disrupt cells can be subjected totreatment with reverse transcriptase and appropriate primers (e.g.,oligo-dT primers) to generate cDNA, and standard nucleic acid extractiontechniques such as those described elsewhere (e.g., Sambrook andRussell, (2001) Molecular Cloning: A Laboratory Manual, Third Edition,Cold Spring Harbor Press) or nucleic acid extraction techniques thatinclude the use of magnetic beads or selective DNA-binding membranes(see, e.g., QIAGEN DNeasy® Blood & Tissue Kit, or Mo Bio PowerFood™Microbial DNA Isolation Kit) can be used. For example, a blood samplecontaining cDNA generated from RNA can be contacted with magnetic beadsthat bind nucleic acid, the beads can be removed, and bound nucleic acidcan be eluted into an appropriate buffer to form a processed sample forfurther analysis using the methods and materials provide herein. Such aprocess can be carried out using a variety of kits including, withoutlimitation, Qiagen BioSprint 96 One-For-All Vet Kit (a rapid andeconomical automated purification of viral nucleic acid and/or bacterialnucleic acid from samples based on magnetic beads) and ChemicellgeneMAG-PCR cleanup kit.

In some cases, a sample treated to disrupt cells and not treated withreverse transcriptase can be subjected to standard RNA extractiontechniques. For example, a blood sample containing RNA can be lysed andeluted into an appropriate buffer to form a processed sample for furtheranalysis using the methods and materials provide herein.

In some cases, a sample can be processed in a manner designed tofragment any nucleic acid present within the sample. For example, largepieces of nucleic acid present within a sample can be subjected to asonication technique and/or restriction digestion (e.g., in casesinvolving samples processed to have double-stranded cDNA) with arestriction endonuclease such as DpnII or CviJI to generate nucleic acidfragments. Such fragmentation can be performed using restrictionendonucleases that are different from those used as recognition oramplifying restriction endonucleases to assess the sample as describedherein.

In some cases, the sample can be treated such that any double-strandednucleic acid (e.g., double-stranded cDNAs) present within the sample isseparated. For example, a biological sample can be heated and thensnap-cooled or can be subjected to chemical (e.g., sodium hydroxide)denaturation. In some cases, when the sample is subjected to a PCR-basedtechnique, certain primer or reaction modifications can be used togenerate preferentially single-stranded product. For example,unidirectional DNA polymerase reactions can be performed with a singlespecific primer. In some cases, the strands of nucleic acid can beseparated, and the strand of interest can be enrichment using specificbiotinylated primers and streptavidin-conjugated magnetic beads. In somecases, selective digestion of one of the strands can be accomplishedusing lambda exonucleases.

As described herein, a sample (e.g., a biological sample) can besubjected to a nucleic acid amplification technique. For example, atissue sample containing extracted nucleic acid can be subjected to aquick PCR-based amplification of one or more specific targets (e.g., 1hour, end-point PCR) or to a whole genome amplification technique (e.g.,Qiagen REPLI-g Screening Kit for high-throughput manual).

Once obtained, a sample to be assessed, whether subjected to a PCR-basednucleic acid technique or not, can be contacted with a probe nucleicacid as described herein. This contacting step can be carried out forany period of time and at any temperature that allows target nucleicacid to hybridize with probe nucleic acid. For example, this step can beperformed between 10 seconds and 24 hours (e.g., between 30 seconds and12 hours, between 30 seconds and 8 hours, between 30 seconds and 4hours, between 30 seconds and 2 hours, between 30 seconds and 1 hour,between 1 minute and 24 hours, between 1 minute and 12 hours, between 1minute and 8 hours, between 1 minute and 4 hours, between 1 minute and 2hours, between 1 minute and 1 hour, between 5 minutes and 1 hour,between 10 minutes and 1 hour, between 15 minutes and 1 hour, or between30 minutes and 1 hour). The initial temperature can be between 15° C.and 100° C. (e.g., between 23° C. and 98° C., between 23° C. and 90° C.,between 23° C. and 85° C., between 23° C. and 75° C., between 23° C. and65° C., between 23° C. and 55° C., between 23° C. and 45° C., between23° C. and 35° C., between 30° C. and 95° C., between 30° C. and 85° C.,between 30° C. and 75° C., between 30° C. and 65° C., between 30° C. and55° C., between 30° C. and 45° C., between 20° C. and 40° C., between20° C. and 30° C., and between 25° C. and 35° C.). The temperatureduring this contacting step can remain constant or can be increased ordecreased. For example, the initial temperature can be between about 40°C. and about 85° C., and then the temperature can be allowed to decreaseto room temperature over a period of about 30 seconds to about 30minutes (e.g., between about 30 seconds and about 15 minutes, betweenabout 30 seconds and about 10 minutes, between about 1 minute and about30 minutes, between about 1 minute and about 15 minutes, or betweenabout 1 minute and about 5 minutes).

Contact of the sample (e.g., a biological sample to be tested) withprobe nucleic acid can occur in the presence of the recognitionrestriction endonucleases, or a separate step of adding the recognitionrestriction endonucleases to the reaction can be performed. Therecognition restriction endonuclease step can be carried out for anyperiod of time and at any temperature that allows the recognitionrestriction endonuclease to cleave recognition restriction endonucleasecut sites formed by the hybridization of target nucleic acid to theprobe nucleic acid. For example, this step can be performed between onesecond and 24 hours (e.g., between one second and 30 minutes, betweenone second and one hour, between five seconds and one hour, between 30seconds and 24 hours, between 30 seconds and 12 hours, between 30seconds and 8 hours, between 30 seconds and 4 hours, between 30 secondsand 2 hours, between 30 seconds and 1 hour, between 1 minute and 24hours, between 1 minute and 12 hours, between 1 minute and 8 hours,between 1 minute and 4 hours, between 1 minute and 2 hours, between 1minute and 1 hour, between 5 minutes and 1 hour, between 10 minutes and1 hour, between 15 minutes and 1 hour, or between 30 minutes and 1hour). The temperature can be between 15° C. and 75° C. (e.g., between15° C. and 75° C., between 15° C. and 65° C., between 15° C. and 55° C.,between 15° C. and 45° C., between 15° C. and 35° C., between 15° C. and30° C., between 23° C. and 55° C., between 23° C. and 45° C., between30° C. and 65° C., between 30° C. and 55° C., between 30° C. and 45° C.,between 30° C. and 40° C., between 35° C. and 40° C., and between 36° C.and 38° C.). Any appropriate concentration of recognition restrictionendonuclease can be used. For example, between about 0.001 units and1000 units (e.g., between about 0.001 units and 750 units, between about0.001 units and 500 units, between about 0.001 units and 250 units,between about 0.001 units and 200 units, between about 0.001 units and150 units, between about 0.001 units and 100 units, between about 0.001units and 50 units, between about 0.001 units and 25 units, betweenabout 0.001 units and 10 units, between about 0.001 units and 1 unit,between about 0.001 units and 0.1 units, between about 0.01 units and1000 units, between about 0.1 units and 1000 units, between about 1 unitand 1000 units, between about 10 units and 1000 units, between about 50units and 1000 units, between about 0.5 units and 100 units, or betweenabout 1 unit and 100 units) of restriction endonuclease can be used.Other restriction endonuclease reaction conditions such as saltconditions can be used according to manufacture's instructions.

When one step of a method provided herein is completed, the resultingreaction product containing cleaved nucleic acid can be used in the nextstep. For example, cleaved nucleic acid of a reaction product can beremoved from uncleaved nucleic acid and used in the next step of themethod. For example, when probe nucleic acid is attached to a solidsupport, the released portions of probe nucleic acid that contain anamplifying restriction endonuclease can be collected and placed incontact with reporter nucleic acid or signal expansion nucleic acid asdescribed herein. The resulting reaction products of a particular stepcan be manually or automatically (e.g., robotically) transferred to alocation containing nucleic acid for the next step (e.g., reporternucleic acid or signal expansion nucleic acid), which nucleic acid canbe attached or not attached to a solid support. In some cases, onereaction of a method described herein can be carried out at one location(e.g., a chamber) of a microfluidic device or blister package device,and the reaction products that are generated can be moved to anotherlocation (e.g., another chamber) that contains nucleic acid for the nextstep (e.g., reporter nucleic acid or signal expansion nucleic acid) viaa channel. In some cases, cleaved nucleic acid of a reaction product canbe used in the next step of the method by removing the uncleaved nucleicacid from the reaction product. For example, when magnetic beads areused as a solid support, a magnetic force can be used to remove themagnetic beads and any attached uncleaved nucleic acid from the reactionproduct. In some cases, two or more reactions of a method providedherein can be carried out at one location (e.g., a single well of amicrotiter plate or a single chamber of a microfluidic device). Forexample, a single compartment can have one region that containsimmobilized probe nucleic acid and another region that containsimmobilized reporter nucleic acid provided that the amplifyingrestriction endonuclease of the immobilized probe nucleic acid is notcapable of cleaving the amplifying restriction endonuclease cut site ofthe reporter nucleic acid unless target nucleic acid hybridizes to theprobe nucleic acid and the recognition restriction endonuclease cleavesthe probe nucleic acid, thereby releasing a portion of the probe nucleicacid that contains the amplifying restriction endonuclease so that it iscapable of cleaving the reporter nucleic acid. In another example, asingle compartment can have one region that contains immobilized probenucleic acid, other regions that contain immobilized signal expansionnucleic acid (e.g., one region that contains a first signal expansionnucleic acid and another region that contains a second signal expansionnucleic acid), and another region that contains immobilized reporternucleic acid provided that the amplifying restriction endonucleases ofimmobilized probe nucleic acid and signal expansion nucleic acid are notcapable of cleaving their intended amplifying restriction endonucleasecut sites until they are released as described herein. Such singlecompartments can be made using partitions or sub-compartments within thesingle compartment. For example, a sample to be tested can be placedinto a single well of a microtiter plate that contains probe nucleicacid, recognition restriction endonucleases, first and second signalexpansion nucleic acid, and reporter nucleic acid such that cleavedreporter nucleic acid and/or signal expansion nucleic acid is producedas described herein when target nucleic acid is present in the samplebeing tested and little or no cleaved reporter nucleic acid and/orsignal expansion nucleic acid is produced when target nucleic acid isnot present in the sample being tested.

Any appropriate method can be used to detect cleaved reporter nucleicacid and/or signal expansion nucleic acid to determine the presence,absence, or amount of target nucleic acid in a sample, which canindicate the presence, absence, or amount of expression of target RNA.For example, size separation techniques can be used to assess reactionproducts for cleaved reporter nucleic acid and/or signal expansionnucleic acid. Examples of such size separation techniques include,without limitation, gel electrophoresis and capillary electrophoresistechniques. In some cases, a melt curve analysis can be performed toassess reaction products for cleaved reporter nucleic acid and/or signalexpansion nucleic acid. As described herein, a label can be used to aidin the detection of cleaved nucleic acid (e.g., reporter nucleic acidand/or signal expansion nucleic acid). Examples of labels that can beused include, without limitation, fluorescent labels (with or withoutthe use of quenchers), dyes, antibodies, radioactive material, enzymes(e.g., horse radish peroxidase, alkaline phosphatase, laccase,galactosidase, or luciferase), redox labels (e.g., ferrocene redoxlabels), metallic particles (e.g., gold nanoparticles), and greenfluorescent protein based labels. For example, the release offluorescently labeled portions of reporter nucleic acid and/or signalexpansion nucleic acid from a solid support can be assessed using commonfluorescent label detectors. In some cases, cleaved reporter nucleicacid and/or signal expansion nucleic acid can be detectedelectrochemically. For electrochemical detection, the reporter nucleicacid and/or signal expansion nucleic acid can include a ferrocene redoxlabel. Reporter nucleic acid and/or signal expansion nucleic acidcontaining ferrocene can be obtained by coupling ferrocene carboxylicacid with an amino-modified oligonucleotide using the carbodiimidereaction in the presence of an excess of ferrocene carboxylic acid. Inone embodiment, for a redox label, such as ferrocene, the detector canbe an electrode for amperometric assay of redox molecules. For example,if the redox label is present in a reduced form of ferrocene, then theelectrode at high electrode potential can provide an oxidation of thereduced form of ferrocene, thereby converting it to an oxidized form offerrocene. The generated current can be proportional to theconcentration of ferrocene label in the solution.

The methods and materials provided herein can be used to assess one ormore samples for target nucleic acid in real-time. For example, afluorescent label/quencher system or an electrochemical redox labelsystem can be used to detect cleavage of reporter nucleic acid and/orsignal expansion nucleic acid in real time.

The methods and materials provided herein can be used to assess one ormore samples (e.g., two, three, four, five, six, seven, eight, nine,ten, 20, 50, 100, 500, 1000, or more) for a single type of targetnucleic acid. For example, 100s of tissue samples (e.g., tissue biopsysamples) can be assessed for expression of a particular target RNA. Insome case, the methods and materials provided herein can be used in amultiplex manner to assess one or more samples for more than one (e.g.,two, three, four, five, six, seven, eight, nine, ten, 20, 50, 100, 500,1000, or more) type of target RNA or target cDNA generated from targetRNA. For example, target nucleic acid for ten different sequences (e.g.,ten different mRNA sequences) can be used to design ten different probenucleic acid molecules. In these cases, each probe nucleic acid can beused in a separate series of reactions within the same device (e.g.,microtiter plate or microfluidic device), and the same label can be usedfor the reporter nucleic acid for each probe nucleic acid. In addition,in some cases, the same amplifying restriction endonuclease can be usedfor each probe nucleic acid, and the same reporter nucleic acid can beused for each reaction series. In some cases, when multiple differentprobe nucleic acid molecules are used in the same reaction series, adifferent reporter nucleic acid having different labels can be used tocorrespond to each probe nucleic acid such that the detected signals canindicate which of the ten target nucleic acids are being detected.

This document also provides kits for performing the methods describedherein. For example, a kit provided herein can include probe nucleicacid with or without being attached to a solid support and/or reporternucleic acid with or without being attached to a solid support. In somecases, such a kit can include a recognition restriction endonuclease,first signal expansion nucleic acid, second signal expansion nucleicacid, or a combination thereof. In some cases, a kit can be configuredinto a microfluidic device that allows for the movement of probe nucleicacid, first signal expansion nucleic acid, second signal expansionnucleic acid, reporter nucleic acid, or recognition restrictionendonucleases (or any combination thereof) as well as a cleaved portionof any such nucleic acid in a manner that allows a detection methodprovided herein to be carried out with or without the nucleic acid beingattached to a solid support. For example, a kit provided herein can be amicrofluidic device capable of receiving a sample and contacting thatsample with probe nucleic acid. The probe nucleic acid can be designedto include a length of nucleotides followed by the sequencecomplementary to the target nucleic acid, which can create a recognitionrestriction endonuclease cut site, followed by an amplifying restrictionendonuclease. The distance from the recognition restriction endonucleasecut site to the amplifying restriction endonuclease can be relativelyshort (e.g., 100, 50, 25, 10, or less nucleotides), while the distancefrom the recognition restriction endonuclease cut site to the beginningof the length of nucleotides can be relatively long (e.g., 50, 100, 150,200, 500, 1000, 2000, or more). In such cases, cleavage of the probenucleic acid at the recognition restriction endonuclease cut site canresult in a relatively small portion that contains the amplifyingrestriction endonuclease and is capable of travelling faster than thelarger uncleaved probe nucleic acid. This difference can allow thecleaved portion containing the amplifying restriction endonuclease toreach an area of the microfluidic device containing signal expansionnucleic acid or reporter nucleic acid so that the next reaction can becarried out without the presence of uncleaved probe nucleic acid. Insome cases, after the smaller portion containing the amplifyingrestriction endonuclease enters the area containing signal expansionnucleic acid or reporter nucleic acid, a valve can be used to preventthe larger uncleaved probe nucleic acid from entering. In some cases, afilter can be used to limit the ability of larger uncleaved probenucleic acid from proceeding to the next reaction location. Similarapproaches can be used during other steps of a method provided herein toseparate cleaved nucleic acid from uncleaved nucleic acid.

In some cases, a kit provided herein can be a portable or self-containeddevice, packet, vessel, or container that can be used, for example, inpoint of care applications. For example, such a kit can be configured toallow a patient or physician's assistant to insert a sample foranalysis. In some cases, a kit can be designed for use in a home settingor any other setting. Once inserted, the sample can be heated (e.g.,heated to about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90,95, or more ° C.) and/or cooled by a heating or cooling mechanismlocated within the kit. For example, an exothermic or endothermicchemical reaction can be initiated within the kit to increase, decrease,or maintain the temperature. Such exothermic or endothermic chemicalreactions can be carried out within the kit without being in fluidcommunication with the reactions of the target nucleic acid detectionmethod. An iron oxidation reaction is an example of an exothermicchemical reaction that can be used to heat a kit provided herein. Anendothermic chemical reaction that can be used to cool a kit providedherein can be a reaction that includes the use of ammonium chloride andwater, potassium chloride and water, or sodium carbonate and ethanoicacid. In general, when detecting DNA target nucleic acid (e.g., targetcDNA generated from target RNA), the kit can be designed to generate, ifneeded, enough heat to denature double stranded DNA present within thesample. The kit also can be designed to generate appropriate heating andcooling temperatures to carry out each step of a detection methodprovided herein. In some cases, a kit provided herein can include atemperature indicator (e.g., color indicator or thermometer) to allows auser to assess temperature.

In some cases, a kit can be designed to provide a user with a “yes” or“no” indication about the presence of target nucleic acid within atested sample. For example, a label having the ability to generate achange in pH can be used, and a visual indicator (e.g., a pH-based colorindicator) can be used to inform the user of the presence of targetnucleic acid based on a change in pH.

In some cases, a point of care or home use device can be designed tocarry out the reactions described herein. For example, point of care orhome use device can be designed to include a series of adjacentchambers. In a relatively simple configuration, for example, a first“sample” chamber can be configured for sample insertion, and can containreagents (e.g., in dry or liquid form) to effect generation of singlestranded nucleic acid fragments. A second “recognition” chamber can beconfigured to receive single stranded nucleic acid fragments from thefirst chamber, and can contain probe nucleic acid and recognitionrestriction endonuclease (e.g., in dry or liquid form). A third“amplification” chamber can be configured to receive cleaved portions ofprobe nucleic acid from the second chamber, and can contain reporternucleic acid (e.g., in dry or liquid form). A fourth “detection” chambercan be configured to receive cleaved portions of marker nucleic acidfrom the third chamber, and can contain a reagent (e.g., in dry orliquid form) that serves as an indicator of whether or not targetnucleic acid was present in the sample. It is noted that one or moreadditional “signal expansion” chambers can be present between the“recognition” chamber and the “amplification” chamber.

In some cases, a point of care or home use device can be configured suchthe chambers are separated from each other by membranes that can providecontrolled passage of reaction materials. For example, chambers can beseparated by membranes that are subject to degradation by particularreagents or solutions. In such cases, a reaction can be confined to aparticular chamber until the membrane separating it from the adjacentchamber degrades, permitting passage of reaction components therebetween.

In some cases, a point of care or home use device can be adapted forautomatic transfer of the reaction mixture between chambers. Forexample, insertion of a sample into the first chamber can trigger areaction or provide a reagent that gradually degrades the membraneseparating the first chamber from the second chamber. Movement of all ora portion of the reaction mixture into the second chamber can in turnprovide a reagent or trigger a reaction that gradually degrades themembrane separating the second chamber from the third chamber. Forexample, if the sample reaction mixture in the first chamber is anaqueous solution, the reagents in the second chamber are dry, and themembrane in the second chamber is degraded by water, movement of theaqueous reaction mixture into the second chamber can trigger degradationof the membrane therein.

In some cases, a point of care or home use device can be adapted forautomatic controlled flow transfer of reaction mixture between chambers.For example, insertion of a sample into the first chamber can trigger areaction or provide a reagent that allows controlled flow movement ofthe sample through absorption media. Movement of all or a portion of thereaction mixture into the second chamber can in turn provide a reagentor trigger a reaction that allows controlled flow movement of the samplethrough absorption media to a third chamber. In such cases, a reactioncan be confined to a particular chamber until the media separating itfrom the adjacent chamber absorbs and permits passage of reactioncomponents there between.

In some cases, a point of care or home use device can be adapted forautomatic controlled flow transfer of reaction mixture between chambers.For example, insertion of a sample into the first chamber can trigger areaction or provide a reagent that allows controlled capillary flowmovement of the sample through micro-fluidic channels. Movement of allor a portion of the reaction mixture into the second chamber can in turnprovide a reagent or trigger a reaction that allows controlled flowmovement of the sample through micro-fluidic channels to a thirdchamber. In such cases, a reaction can be confined to a particularchamber until the microfluidic channel permits passage of reactioncomponents there between.

In some cases, a point of care or home use device can be adapted forautomatic controlled flow transfer of reaction mixture without chambers.For example, insertion of a sample into the device can trigger areaction or provide a reagent that allows controlled capillary flowmovement of the sample through microfluidic channels. Movement of all ora portion of the reaction mixture in the microfluidic channel cantrigger a reaction that allows reagents to enter the reaction mixture ina continuous flow-through manner with no specific chamber for areaction. In such cases, a reaction does not need to be confined to aparticular section of the microfluidic channel.

In some cases, transfer of a reaction mixture from one chamber to thenext can be controlled by a user. An exemplary user-controlled,pen-style point of care or home use device is depicted in FIG. 7. Device300 can include sample collector 310 and reaction unit 320. Samplecollector 310 can have cap 312 with screw threads 314, shaft 316, andswabber 318. Swabber 318 can be smooth or rough, and in some cases canhave bristles (e.g., smooth or rough bristles) or a matted texture tofacilitate sample collection from, for example, the inside cheek,throat, or skin of an individual to be tested.

Reaction unit 320 can include tube 322, open end 324 reversibly closedby safety cap 326, and closed end 328. Open end 324 can have internalscrew threads, and cap 326 can have external screw threads 329. Screwthreads 329 of safety cap 326, as well as screw threads 314 of samplecollector cap 312, can be adapted to mate with the internal screwthreads at open end 324, such that either safety cap 326 or samplecollector 310 can be screwed into open end 324.

Tube 322 can contain several chambers, such as lysing and isolationchamber 330, recognition and amplification chamber 360, and detectionchamber 390. As described herein, the chambers can be separated from oneanother to prevent premature mixing of reaction components. Tube 322 andthe chambers contained therein can be made from, for example, clearplastic (e.g., polycarbonate, acrylic, nylon, or PVC). Tube 322 also cancontain first and second safety bands 340 and 370, and first and secondspring returns 350 and 380.

Lysing and isolation chamber 330 can be positioned proximal to open end324. Lysing and isolation chamber 330 can have proximal end 332, distalend 334, proximal membrane 336, distal membrane 337, and reactioncompletion indicator 338. Proximal membrane 336 can be located adjacentto proximal end 332, and distal membrane 337 can be located adjacent todistal end 334. Membranes 336 and 337 can be made from, for example,synthetic rubber, natural latex rubber, or silicone. Chamber 330 cancontain reagents for lysing cells as well as reagents for cleaving anddenaturing cellular nucleic acids. Reaction completion indicator 338 canbe, for example, a built in timer or stop watch, a built in pHindicator, a built in color change reagent, or a conductivity probe, andcan indicate when cell lysis and nucleic acid sample generation aresufficient to proceed to the next step.

First safety band 340 can be positioned distal to lysing and isolationchamber 330 within tube 322, and first spring return 350 can bepositioned distal to first safety band 340. First safety band 340 canbe, for example, connected to a tab or strap, and can be moved orremoved from reaction unit 320 by pulling on the tab or strap. Firstspring return 350 can be made from a shape memory material that can becompressed and then automatically return to or toward its originalconfiguration.

The safety band 340 can be attached to the tube as a secured ring thatcan be, for example, over molded as a soft rubber component or insertedas a spring like split ring component. The safety band 340 can lock theposition of the lysing and isolation tube chamber 340, preventing linearsliding of the lysing and isolation chamber 330 to that of therecognition and amplification chamber 360. Upon removal of safety band340, the user can actuate linear movement of the entire device 300 byholding the proximal end firm and pressing the distal closed end 328such that both distal chambers recognition and amplification 360 anddetection chamber 390 are moved toward the lysing and isolation chamber330. The needle and sample collector 362 can pierce membrane 337 andenter the lysing and isolation chamber 330. The user can release a firmhold on the assembly and spring return 350 can draw the sample intorecognition and amplification chamber 360. After completion of thereaction, the user can remove safety band 370, and the user can actuatelinear movement of the assembly by holding the recognition andamplification chamber 360 firm and pressing the distal closed end 328such that the detection chamber 390 moves toward the recognition andamplification chamber 360. The needle and sample collector 392 canpierce membrane 366. The user can release the firm hold on the assembly,and spring return 380 can draw the sample into detection chamber 390.

Recognition and amplification chamber 360 can be positioned distal tofirst spring return 350. Chamber 360 can have proximal end 361, which inturn can have piercing needle and sample collector 362, distal end 364,membrane 366, and reaction completion indicator 368. Recognition andamplification chamber 360 can contain, for example, probe nucleic acidand reporter nucleic acid and restriction endonucleases for use inenzymatic amplification cascades as described herein. Piercing needleand sample collector 362 can have a pointed, beveled, or barbed tip. Inaddition, the interior of piercing needle and sample collector 362 canbe in fluid communication with the interior of recognition andamplification chamber 360, such that a nucleic acid test sample can becollected from lysing and isolation chamber 330 and transferred torecognition and amplification chamber 360 via collector 362. Membrane364 can be located adjacent to distal end 364, and can be made from, forexample, synthetic rubber, natural latex rubber, or silicone. Reactioncompletion indicator 368 can be, for example, a built in timer or stopwatch, a built in pH indicator, a built in color change reagent, or aconductivity probe, and can indicate when cell lysis and nucleic acidsample generation are sufficient to proceed to the next step.

Second safety band 370 can be positioned distal to recognition andamplification chamber 360 within tube 322, and second spring return 380can be positioned distal to second safety band 370. Second safety band370 can be, for example, connected to a tab or strap, and can be movedor removed from reaction unit 320 by pulling on the tab or strap. Secondspring return 380 can be made from a shape memory material (e.g., springsteel, plastic, or rubber) that can be compressed and then automaticallyreturn to or toward its original configuration.

Detection chamber 390 can be positioned distal to second spring return380, adjacent to closed end 328 of tube 322. Detection chamber 390 canhave proximal end 391, which in turn can have piercing needle and samplecollector 392, and distal end 394. Piercing needle and sample collector392 can have a pointed, beveled, or barbed tip. In addition, theinterior of piercing needle and sample collector 392 can be in fluidcommunication with the interior of detection chamber 390, such that areaction sample can be collected from recognition and amplificationchamber 360 and transferred to detection chamber 390 via collector 392.Detection chamber 390 can contain a substrate for an enzyme marker suchas a substrate for horseradish peroxidase (HRP) (e.g., ABTS, TMB, OPD)or alkaline phosphatase (AP) (e.g., PNNP).

Sample collector 310 and reaction unit 320 can be packaged together andsold as a kit. In use, the sample collector 310 can be removed from thepackage, and a swab can be obtained from, for example, a subject's body.Cap 326 can be removed from open end 324 of tube 322, and samplecollector 310 can be screwed into open end 324 such that all or aportion of swabber 318 extends through proximal membrane 336 and intothe interior of lysing and isolation chamber 330. The sample can bemixed (e.g., by shaking), and the lysing and nucleic acid preparationcan proceed for a particular length of time, or until reactioncompletion indicator 338 indicates that the user can proceed to the nextreaction step.

When the nucleic acid sample is ready, the user can remove first safetyband 350 from reaction unit 320, and can actuate reaction unit 320 suchthat piercing needle and sample collector 362 moves proximally topenetrate distal membrane 337 of lysing and isolation chamber 330,collects a sample from chamber 330, and, by virtue of first springreturn 350, moves distally to its original position. The sample canagain be mixed, and the recognition and amplification steps can proceedfor a particular length of time, or until reaction completion indicator368 indicates that the user can proceed to the next reaction step.

When the reaction sample is ready, the user can remove second safetyband 380 from reaction unit 320, and can actuate reaction unit 320 suchthat piercing needle and sample collector 392 moves proximally topenetrate membrane 366 of recognition and amplification chamber 360,collects a sample from chamber 360, and, by virtue of second springreturn 380, moves distally to its original position. The sample canagain be mixed, and marker released during the amplification step can bedetected (e.g., colorimetrically or fluorescently). In some cases, theouter surface of tube 322 can have a color code printed thereon, so auser can compare the color of detection chamber 390 with the color codeto determine whether or not the tested sample contains target nucleicacid.

Device 300 can have any suitable dimensions. For example, the size ofdevice 300 can approximate that of a pen or a marker, which can make itparticularly convenient to transport. In some cases, device 300 can havea diameter at its widest point of about 0.25 to about 2 cm (e.g., 0.25,0.3, 0.4, 0.5, 0.6, 0.75, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, or 2 cm), and a length of about 5 cm to about 200 cm(e.g., 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, or 200 cm).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Formation and Cleavage of Target-Probe Hybrids

An oligonucleotide probe (5′-thiol-GGT AGT GCG AAA TGC CAT TGC TAG TTGTTT-biotin-3′; SEQ ID NO:1) that was modified with a thiol group at the5′ end and a biotin molecule at the 3′ end was conjugated to horseradishperoxidase (HRP). Conjugation was performed using the SMCC reagentaccording to a technique modified from Dill et al. (Biosensors andBioelectronics, 20:736-742 (2004)). The HRP conjugate solution wasincubated with a streptavidin-coated ELISA plate to immobilize theHRP-oligonucleotide probe to the surface via a biotin-streptavidininteraction. The ELISA plate was then incubated with differentconcentrations of a target oligonucleotide (5′-AAA CAA CTA GCA ATG GCATTT-3′; SEQ ID NO:2). The target oligonucleotide sequence wasreverse-complementary to the probe sequence to form a double-strandedhybrid molecule. After washing, the plate was incubated in a solutioncontaining the restriction endonuclease BfaI. BfaI specificallyrecognizes the sequence 5′-CTAG-3′ and cleaves the double-stranded,target-probe hybrids to release the HRP-oligonucleotide into thereaction solution. After a two-hour incubation at 37° C., the reactionsolution was transferred to a new ELISA plate. The cleavedHRP-oligonucleotide was contacted to 3,3′,5,5′-tetramethyl benzidine(TMB) to form a colored reaction product.

When the restriction endonuclease BfaI was added in excess to thereaction mixture, a clear direct dependence between the amount ofreleased HRP-probe and the concentration of oligonucleotide target wasobserved (FIG. 6A). The detectable target concentration wasapproximately 1 nM. This detection limit was obtained by directmeasurement without any secondary signal amplification. The addition ofa restriction endonuclease signal amplification cascade as describedherein can further improve the detection limit by several orders ofmagnitude.

When the HRP-oligonucleotide probes were pre-incubated with an excess oftarget oligonucleotide (500 nM), the amount of cleavedHRP-oligonucleotide probe was limited by the amount of recognitionrestriction endonuclease BfaI (FIG. 6B). Taken together, these datademonstrate that recognition restriction endonucleases can be used toinitiate the restriction endonuclease cascades described herein.

Example 2 Detecting Target Nucleic Acid using Probe Nucleic Acid andReporter Nucleic Acid

A target nucleic acid is selected. Once selected, target nucleic acid isanalyzed using a common genetic database such as GenBank® and/or acomputer-based sequence analysis program to identify a portion of thetarget nucleic acid that contains a cut site for a restrictionendonuclease. Probe nucleic acid is designed to be complementary to atleast a portion of target nucleic acid that contains a cut site. Oncedesigned and obtained by standard oligonucleotide synthesis methods,probe nucleic acid is conjugated to an amplifying restrictionendonuclease and immobilized to the surface of a first well of amicrotiter plate. A sample to be tested is incubated in the first well.If target nucleic acid is present in the sample, at least a portion ofthe target nucleic acid hybridizes to the probe nucleic acid, andthereby forms a recognition restriction endonuclease cut site. Therecognition restriction endonuclease is added to the first well havingthe sample and probe nucleic acid. The microtiter plate is incubated at37° C. for an appropriate length of time for the cleavage reaction toproceed.

Upon cleavage of probe nucleic acid by the recognition restrictionendonuclease, the reaction solution containing the released portion ofthe probe nucleic acid is transferred into a second well. The secondwell contains reporter nucleic acid that is immobilized to the surfaceand contains at least one double-stranded portion having an amplifyingrestriction endonuclease cut site. Reporter nucleic acid also has afluorescent label. Upon transfer to the second chamber, the amplifyingrestriction endonuclease bound to the released portion of the probenucleic acid contacts the reporter nucleic acid. The amplifyingrestriction endonuclease cleaves reporter nucleic acid at thedouble-stranded amplifying restriction endonuclease cut site to form atleast two portions. The liberated portion of the reporter nucleic acidhaving the fluorescent label is moved to a third microtiter plate well,and a standard fluorescent reader is used to measure any fluorescentsignal.

A standard curve of known amounts of target nucleic acid is used toquantify the amount of target nucleic acid in the tested sample.

Example 3 Detecting Target Nucleic Acid using Probe Nucleic Acid, FirstSignal Expansion Nucleic Acid, Second Signal Expansion Nucleic Acid, andReporter Nucleic Acid

Once selected, target nucleic acid is analyzed using a common geneticdatabase such as GenBank® and/or a computer-based sequence analysisprogram to identify a portion of target nucleic acid that contains a cutsite for a restriction endonuclease. Probe nucleic acid is designedbased on the desired target nucleic acid as described herein. Standardoligonucleotide synthesis methods are used to make the probe nucleicacid, which is then conjugated to an initial amplifying restrictionendonuclease and immobilized to the surface of a first well of amicrotiter plate. A sample to be tested for the target nucleic acid isincubated in the first well. If target nucleic acid is present in thesample, at least a portion of target nucleic acid hybridizes to probenucleic acid and thereby forms a recognition restriction endonucleasecut site. Recognition restriction endonuclease is added to the firstwell having the sample and probe nucleic acid. The microtiter plate isincubated at 37° C. for an appropriate length of time for the cleavagereaction to proceed.

After cleavage of the probe nucleic acid:target nucleic acid hybrid bythe recognition restriction endonuclease, the reaction solutioncontaining the free portion of probe nucleic acid is transferred toanother well that includes first signal expansion nucleic acid andsecond signal expansion nucleic acid. The first signal expansion nucleicacid and second signal expansion nucleic acid creates a positivefeedback loop that causes an exponential acceleration of release ofinitial amplifying restriction enzymes. The reaction product from thiswell is transferred to another well containing reporter nucleic acid,and cleavage of the reporter nucleic acid is used to determine thepresence, absence, or amount of target nucleic acid in the sample. Astandard curve of known amounts of target nucleic acid is used toquantify the amount of target nucleic acid in the tested sample.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for assessing RNA expression, saidmethod comprising: (a) contacting a sample to be assessed for expressionof a target RNA with a probe nucleic acid comprising an amplifyingrestriction endonuclease and a nucleotide sequence complementary to asequence of said target RNA or a cDNA of said target RNA underconditions wherein, if said target RNA or said cDNA is present in saidsample, at least a portion of said target RNA or said cDNA hybridizes toat least a portion of said probe nucleic acid to form a double-strandedportion of nucleic acid comprising a restriction endonuclease cut site,(b) contacting said double-stranded portion of nucleic acid with arecognition restriction endonuclease having the ability to cut saiddouble-stranded portion of nucleic acid at said restriction endonucleasecut site under conditions wherein said recognition restrictionendonuclease cleaves said double-stranded portion of nucleic acid atsaid restriction endonuclease cut site, thereby separating a portion ofsaid probe nucleic acid comprising said amplifying restrictionendonuclease from at least another portion of said probe nucleic acid,(c) contacting said portion of said probe nucleic acid comprising saidamplifying restriction endonuclease with a reporter nucleic acidcomprising a double-stranded portion of nucleic acid comprising arestriction endonuclease cut site of said amplifying restrictionendonuclease under conditions wherein said amplifying restrictionendonuclease cleaves said reporter nucleic acid at said restrictionendonuclease cut site of said amplifying restriction endonuclease,thereby separating a portion of said reporter nucleic acid from at leastanother portion of said reporter nucleic acid, and (d) determining thepresence or absence of said portion of said reporter nucleic acid,wherein the presence of said portion of said reporter nucleic acidindicates that said sample contains said target RNA or said cDNA,thereby indicating expression of said target RNA, and wherein theabsence of said portion of said reporter nucleic acid indicates thatsaid sample does not contain said target RNA or said cDNA, therebyindicating a lack of expression of said target RNA.